WO2007134328A2 - Geldanamycins and their quinone moieties inhibit cancer by acting on mitochondrial voltage-dependent anion channel (vdac) protein - Google Patents

Geldanamycins and their quinone moieties inhibit cancer by acting on mitochondrial voltage-dependent anion channel (vdac) protein Download PDF

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WO2007134328A2
WO2007134328A2 PCT/US2007/068988 US2007068988W WO2007134328A2 WO 2007134328 A2 WO2007134328 A2 WO 2007134328A2 US 2007068988 W US2007068988 W US 2007068988W WO 2007134328 A2 WO2007134328 A2 WO 2007134328A2
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compound
cells
tumor
cell
inhibition
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WO2007134328A3 (en
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Qian Xie
David Wenkert
Yuchai Shen
George F. Vande Woude
Rick Hay
Robert Wondergem
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Van Andel Research Institute
Michigan State University
East Tennessee State University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/13—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups
    • C07C205/20—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups having nitro groups and hydroxy groups bound to carbon atoms of six-membered aromatic rings
    • C07C205/21—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups having nitro groups and hydroxy groups bound to carbon atoms of six-membered aromatic rings having nitro groups and hydroxy groups bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C205/22—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by hydroxy groups having nitro groups and hydroxy groups bound to carbon atoms of six-membered aromatic rings having nitro groups and hydroxy groups bound to carbon atoms of the same non-condensed six-membered aromatic ring having one nitro groups bound to the ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/27—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups
    • C07C205/35—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups having nitro groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C205/36—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups having nitro groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton to carbon atoms of the same non-condensed six-membered aromatic ring or to carbon atoms of six-membered aromatic rings being part of the same condensed ring system
    • C07C205/37—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups having nitro groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton to carbon atoms of the same non-condensed six-membered aromatic ring or to carbon atoms of six-membered aromatic rings being part of the same condensed ring system the oxygen atom of at least one of the etherified hydroxy groups being further bound to an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/41—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a ring other than a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D203/00—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D203/04—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D203/06—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D203/08—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring nitrogen atom
    • C07D203/14—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring nitrogen atom with carbocyclic rings directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D225/00—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom
    • C07D225/04—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D225/06—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems

Definitions

  • the present invention in the field of cancer pharmacology is directed to quinone and hydroquinone chemical moieties present within geldanamycin and its derivatives), that inhibit cancer cell activities at nanomolar concentrations and act at the outer membrane mitochondrial voltage-dependent anion channel (VDAC) protein. These compounds are used to inhibit HGF-dependent, Met-mediated tumor cell activation, growth, invasion, and metastasis and serve as potent anticancer agents.
  • VDAC membrane mitochondrial voltage-dependent anion channel
  • G is a benzoquinone ansamycin antibiotic.
  • GA derivatives are in clinical trials as anticancer drugs.
  • L. Whitesell et al. Proc Natl Acad Sci USA91, 8324 (1994) discovered that the molecular chaperone, HSP90, is a target for GA.
  • GA binds to the HSP90 binding pocket, blocking ATP hydrolysis which is required for HSP90 chaperone function.
  • GA-induced inactivation of HSP90 function results in the degradation of a large set of oncoproteins, thereby accounting for its therapeutic action.
  • These drugs target HSP90 chaperone activity at nanomolar concentrations.
  • the outer membrane mitochondrial voltage-dependent anion channel (VDAC) protein is a component of the mitochondrial permeability transition pore (PTP), a calcium-, voltage-, and pH-sensitive pore that opens when calcium levels increase, especially when accompanied by oxidative stress (DR Green et al., Science 281, 1309 (1998). I. Marzo et al., J Exp Med 187, 1261 (1998)).
  • PTP mitochondrial permeability transition pore
  • the present invention is directed to the targeting of VDAC by GA and derivatives, with an emphasis on this activity mediated by redox-cycling benzoquinone/ semiquinone/ hydroquinone moieties of GA or its derivatives.
  • the present invention identifies VDAC as a novel GA binding protein and shows that GA inhibits membrane current at pM levels.
  • ubiquinone 0 and decyl- ubiquinone known benzoquinone inhibitors of mitochondrial PTP function that act like GA, also inhibit HGF/SF induced urokinase (uPA) plasmin protease activation as well as action currents measured in whole-cell voltage clamp assays with GA blocking at pM levels.
  • uPA HGF/SF induced urokinase
  • a benzoquinone moiety of a novel aziridinyl derivative of GA has nanomolar inhibitory activity against HGF/SF induced uPA activity identifying this compound as a useful agent against cancer.
  • the present invention is directed to a redox-cycling benzoquinone, semiquinone or hydroquinone compound, or a pharmaceutically acceptable salt of the benzoquinone , which compound has the property of inhibiting (a) the activation of Met by HGF/SF in cancer cells and (b) HGF/SF-induced tumor cell invasion, which benzoquinone, semiquinone, hydroquinone or compound or salt binds to and inhibits the activity of mitochondrial VDAC protein of cells.
  • the above benzoquinone is preferably one that is present in the structure of GA or a 17-alkylamino 17-demethoxy derivative of GA.
  • the above compound inhibits VDAC to an extent that is equal to or exceeds the inhibitory action of mitochondrial specific VDAC inhibitors ubiquinone (UbO) or decyl- ubiquinone (Decyl-Ub).
  • the above compound preferably inhibits non-selective cation currents in the cells at subnanomolar concentrations, including at concentrations below ⁇ 10 "10 M.
  • the above compound preferably inhibits Ca 2+ influx, thereby acting as a calcium channel blocker.
  • the compound is preferably selected from the group consisting of:
  • CMQ 5 -Acetamido-2-carboxymethylamino-3 -methylbenzoquinone
  • a most preferred compound is ARQ.
  • a pharmaceutical compositions comprising (a) any of the above compounds; and (b) a pharmaceutically acceptable carrier or excipient.
  • Also included herein is a method of inhibiting function of mitochondrial VDAC protein and thereby inhibiting cation flux in a cell, comprising providing to the cell an effective amount of
  • geldanamycin or a derivative thereof, or (ii) a redox-cycling benzoquinone/semiquinone/or hydroquinone moiety of GA or derivative,
  • Also included is a method of inhibiting a HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell comprising providing to the cells an effective amount of the compound or pharmaceutical composition as above, wherein the compound has an IC50 of less than about 10 "7 M, less than about 10 "8 M, or preferably less than about 10 "9 M, for inhibition of the biological activity.
  • Such biological activity may be the induction of uPA activity in the cells, or growth, scatter or invasion of the cells, wherein such invasion is of the type that results in tumor metastasis.
  • a method for blocking calcium channels or cation flux in a cell comprises providing to the cells an effective amount of a compound or pharmaceutical composition thereof, which inhibits the activity of the mitochondrial VDAC protein which comprises providing to the cell an effective amount of a compound that is (i) geldanamycin (GA) or (ii) a derivative thereof, or (iii) a redox-cycling benzoquinone, semiquinone or /hydroquinone moiety of the GA or derivative, or (iv) a pharmaceutically acceptable salt of the GA, derivative or moiety, or a pharmaceutical composition that comprises any of (i) - (iv), which compound binds to and inhibits VDAC function and cation flux at subnanomolar concentrations.
  • a compound or pharmaceutical composition thereof which inhibits the activity of the mitochondrial VDAC protein which comprises providing to the cell an effective amount of a compound that is (i) geldanamycin (GA) or (ii) a derivative thereof, or (iii) a redox-
  • the compound is preferably one disclosed above.
  • the cell is preferably a human cell or the subject is a human.
  • the invention is also useful in treating a non-human animal in the context of veterinary medicine.
  • Figure 1A-1D show that VDAC binds to Geldanamycin-conjugated affinity beads.
  • Fig. IA GA-conjugated affinity beads were used in pull-down experiments with cell lysates from MDCK (left panel) and DBTRG cultures (right panel). Cells were treated with either 1 ⁇ M GA, 17-AAG, or under basal conditions for 24 h prior to harvest and then lysed. An aliquot of each cell lysate was incubated with GA-conjugated affinity beads. Eluates from the beads were analyzed by SDS-PAGE followed by Coomassie blue staining of the gel. Stained protein bands were excised for analysis by mass spectrometry (Table 1). Lane 1 : Molecular weight markers. Lanes 2 and 3: Total cell lysates precipitated by control and GA-conjugated affinity beads, respectively.
  • Lanes 4 and 5 GA-conjugated affinity bead pull-downs of cell lysates from cells treated for 24 h with 1 ⁇ M 17-AAG and GA, respectively.
  • the assignments of individual HSP90 and VDAC bands were established by mass spectrometry. Other bands identified by mass spectrometry are actins or tubulins, which have been reported as HSP90 substrates (JN Oa ⁇ seid et al., MolBiol Cell 5, 1265 (1994)).
  • Fig. IB GA-conjugated affinity bead pull-downs of HSP90 and VDAC from washed isolated mitochondria.
  • Lanes 1-3 are Western blot analyses of the GA affinity bead pull-down pellet fractions of the post-nuclear cell lysate (Sl) (lane 1), the mitochondrial wash supernatant (S2) (Lane T), and solubilized mitochondrial fractions (P3) (Lane 3).
  • Lanes 4-6 are aliquots of supernatants after GA affinity bead pull-downs of Sl, S2, and solubilized P3.
  • HSP90 upper half
  • VDAC lower half
  • Both HSP90 ⁇ and VDAC were pulled down by GA affinity beads from the post-nuclear total MDCK and DBTRG cell lysate (lane 1). Only VDAC was pulled down from the first mitochondrial supernatant wash and less HSP90 was not detected after washing (lane X). HSP90 (and less VDAC) remains in the supernatant fractions after the GA-bead pull-down (lanes 4-6). VDAC enrichment occurs in the mitochondrial fraction.
  • Fig. 1C Free GA release of HSP90 from GA-coupled beads.
  • GA-affmity beads were incubated with MDCK whole cell lysates at 4 0 C for 4 h. After the beads were washed 3 times with TNSEV buffer, GA was added at the indicated concentrations for 1 h at 4 0 C to release HSP90 and VDAC. The HSP90 and VDAC remaining on the beads were detected following the 1-h incubation by immunoblotting as described above. GA can release HSP90 from the beads after a 1-h incubation, while VDAC doesn't show any release.
  • Fig. ID Free GA release of VDAC from GA-coupled beads. Mitochondria purification from MDCK cells and DBTRG cells was performed by differential centrifugation (see Materials and Methods).
  • rhVDAC recombinant human VDAC
  • Figure 2 shows that VDAC and "permeability transition pore" inhibitors UbO, Decyl-Ub, H2DIDS, and CsA block HGF/SF mediated uPA-plasmin activation.
  • MDCK cells were seeded in 96-well plates at 1500 cells/well. 24 hrs later, HGF/SF (60 ⁇ g/ml) was added to all wells (with the exception of wells used to calculate basal plasmin activation). Immediately after HGF/SF addition, compounds at different concentrations were added into wells and after another 24 hours incubation, plates were washed and 200 ml of reaction buffer were added to each well.
  • CsA and H 2 DIDS showed activity to 100 ⁇ M, while UbO and Decyl-Ub block uPA-plasmin activation at 10 ⁇ M and 100 ⁇ M, respectively.
  • Figures 3A-3D show effects of the benzoquinone 17-ARQ (Figs 3 A and 3B) and the fluorinated GA derivative, 17-FEG (Figs. 3C and 3D) on HGF-stimulated biological activities in MDCK cells.
  • Fig. 3 A shows Western blots of cell lysates from MDCK incubated with various dilutions of the benzoquinone 17-ARQ in DMSO at concentrations ranging from 10 "6 to 10 "12 M and incubated for 24 hours .
  • Control cells (lane 1, labeled "C") were not treated; cells in lane 2, labeled "D” were treated with the DMSO solvent only. Cells were then lysed, and processed for Western blots.
  • Protein concentration was determined by DC protein assay (Bio-Rad). Equal quantities of protein were loaded onto gels and separated by SDS-PAGE. Proteins were transferred to PVDF membranes as shown above for Western blotting. The membranes were blocked and blotted with specific antibodies as noted above. Membranes were processed as described in the Examples. Protein bands reactive with specific antibodies for Met, HSP90, VDAC and ⁇ actin (loading control) are shown.
  • Fig. 3B shows that ARQ inhibits uPA-plasmin activation induced by HGF in a dose dependent manner over the range of 10 "5 to 10 "8 M. Controls were either untreated cells or cells treated with the DMSO solvent. The assay is described in Example I. Fig.
  • FIG. 3C shows results of treating MDCK cells with 17-FEG serially diluted with DMSO at concentrations from 10 "6 M to 10 "9 M, respectively. Incubation and processing was as above. Control (C) cells were not treated (lane 1) and additional controls (D) were treated with DMSO only (lane 2). Western blots of proteins from lysed cells were prepared as described herein. Antibodies used are those shown given above for Met, HSP90, VDAC and ⁇ -actin (loading control).
  • Fig. 3D shows results of a uPA activity assay conducted as described in the Examples and above for Fig. 3B. Cells were exposed to HGF/SF (100ng/ml) alone or with 17-FEG at the indicated concentrations.
  • FIG. 4A shows effects of UbO, Decyl-Ub, and GA on all external cations on whole-cell current in MDCK cells treated with HGF/SF.
  • Fig. 4A Membrane current as a function of 300-ms voltage ramps (I-V plots) from -100 to 100 mV.
  • NMDG + indicates substitution of all external cations with n-methyl-D-glucamine.
  • Fig. 4B shows that
  • Membrane current recorded at -80 mV ( • ) and 80 mV ( o )versus time. Numerals indicate respective times corresponding to the numbered plots of current vs. voltage (I-V's) shown in Fig. 4A. NMDG + as in Fig. 4A. Holding potential -30 mV. Fig. 4C. Effect of ubiquinone. Membrane current as a function of 300-ms voltage ramps (I-V plots) from -100 to 100 mV. Cs aspartate solution in pipette. Fig. 4D. Membrane current recorded at -80 mV ( • ) and 80 mV ( o )versus time.
  • Fig. 4E Effect of decyl-ubiquinone. Membrane current as a function of 300-ms voltage ramps from -100 to 100 mV. Cs aspartate solution in pipette.
  • Fig. 4F Membrane current recorded at -80 mV ( • ) and 80 mV ( o )versus time.
  • Numerals indicate respective times corresponding to the numbered plots of current vs. voltage (I-V's) shown in Fig. 4E.
  • Holding potential -30 mV.
  • Fig. 4G Effect of geldanamycin. Membrane current as a function of 300-ms voltage ramps from -90 to 90 mV. K gluconate solution in pipette.
  • Figure 5 shows the chemical/schematic structure of four types of affinity beads to which different molecules have been conjugated for use in cell lysate pull-down experiments: C 6 -GA-beads, C12-GA beads, PEG-GA beads and C 6 -AQ beads.
  • Figure 6 shows results of an experiment in which MDCK cell lysates were pulled down with each of the four benzoquinone bead preparations in Fig. 5.
  • Samples were analyzed by SDS PAGE WB for HSP90 (upper panel) and VDAC (lower panel).
  • Lane 1 conventional C 6 -GA beads; lane 2, Ci 2 -GA beads; lane 3, PEG-GA beads; and lane 4, C 6 - AQ beads. All beads with GA effectively pulled down VDAC (only C 6 -AQ, a benzoquinone lacking the ansamycin ring, did not pull-down VDAC.
  • Figure 7/1 - 7/3 is a list of GA and derivatives and benzoquinone compounds of the present invention showing chemical structures. Figs. 7/1 and 7/2 show GA and derivatives compounds (given numeric designations). Fig. 7/3 shows benzoquinone compounds derived from some of these GA compounds (given lower case letter designations). DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the compounds useful in this invention have the property of inhibiting (a) the activation of Met by HGF/SF in cancer cells and (b) HGF/SF-induced tumor cell invasion.
  • the GA and preferred derivatives or salts inhibit HSP90, but also may inhibit mitochondrial activity through VDAC as well as inhibiting non-selective cation currents in cells at picomolar concentrations.
  • the compounds preferably have an IC50 of less than about 10 ⁇ 8 M for inhibition of biological activity.
  • Compounds that are useful in the above methods preferably are those in which VDAC inhibition equals or exceeds that of mitochondria specific ubiquinone (UbO) or decyl-ubiquinone (Decyl-Ub).
  • Nonlimiting examples of useful benzoquinone compounds (or their salts) that make up part of the ring structure of GA and its derivatives were listed above.
  • the present invention is directed to a method for inhibiting, among other activities, HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell.
  • Met-mediated biological activities include the induction of uPA activity in the cells or cell growth, scatter or invasion.
  • invasive activity reflects the cells' ability to metastasize in vivo.
  • the present methods include the inhibition of tumor metastasis of a primary or recurrent tumor in a subject
  • the inhibition produced by the present compounds and methods results in measurable regression of the primary, recurrent or metastatic tumor or measurable attenuation of tumor growth in the subject.
  • the method is particularly useful for the treatment of a Met-positive tumors in a susceptible or affected subject.
  • the compound or pharmaceutical composition is administered to a subject who is either (a) at risk for development of the tumor, or (b) in the case of an already treated subject, at risk for recurrence of the tumor.
  • the above method is also used to induce an antitumor or anticancer response in a mammal, preferably a human, which response is: (a) a partial response characterized by (i) at least a 50% decrease in the sum of the products of maximal perpendicular diameters of all measurable lesions; (ii) no evidence of new lesions, and (iii) no progression of any preexisting lesions, or (b) a complete response characterized by the disappearance of all evidence of tumor or cancer disease for at least one month.
  • the invention is directed to a method of inhibiting a HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell, comprising providing to the cells an effective amount of a compound as described herein that has an IC50 of less than about 10 "8 M or less than about 10 "9 M or less than about 10 "10 M or less than about 10 "11 M.
  • the biological activity may be measured the induction of uPA activity in the cells, or patch clamp cell membrane cationic current, scatter of the cells, invasion of said cells in vitro.
  • a pharmaceutical composition according to this invention comprises the compounds described herein in a formulation that, as such, is known in the art.
  • compositions within the scope of this invention include all compositions wherein the GA derivative, preferably a benzoquinone, a semiquinone or a hydroquinone salt of the benzoquinone is contained in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 mg to 100 mg/kg/body mass, more preferably 1 mg to 100 mg/kg body mass, more preferably 10 mg - 10 mg/kg body mass.
  • a preferred dose of a benzoquinone or other compound of the present invention is one that is sufficient to inhibit VDAC activity, for example, a compound that has an IC50 of less than about 10 "8 M for inhibition of a biological activity mediated by VDAC activity in mitochondria and cation flux in a cell..
  • Preferred doses are lower than doses that are typically associated with inhibition of HSP90 ( Figure 3 C & D).
  • the effective does is preferably one that inhibits uPA (Figure 3 D) and cation flux in a cell ( Figure 4A-4H) and interferes with growth, invasion, scatter, etc, of such a cell.
  • geldanamycin drug effect on HSP90 is measured by HSP90 induction and reduction of Met ( Figure 3C).
  • the effectiveness of the compound or pharmaceutical composition may be measured as its biological activity in an assay as described herein, in vitro or in vivo.
  • the biological effectiveness may be measured by direct testing of the target parameters of the compound in the treatment of cancer in a subject, such as in any known and accepted murine or other small or large animal model, or in a human subject being treated.
  • the effective dose or amount may be less than the amount needed to cause significant inhibition, such as >90% or >80% or >70% or >60% or >50% inhibition or of HSP90 or of a biological effect mediated by HSP90 but not one mediated by VDAC or by mechanisms that work through mitochondrial function or through inhibition of cation flux in a cell.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically as is well known in the art.
  • suitable solutions for administration by injection or orally may contain from about 0.01 to 99 percent, active compound(s) together with the excipient.
  • the pharmaceutical preparations of the present invention are manufactured in a manner which is known, for example, by means of conventional mixing, granulating, dissolving, or lyophilizing processes.
  • Suitable excipients may include fillers binders, disintegrating agents, auxiliaries and stabilizers, all of which are known in the art.
  • Suitable formulations for parenteral administration include aqueous solutions of the proteins in water-soluble form, for example, water-soluble salts.
  • Compounds are preferably be dissolved in dimethylsulfoxide (DMSO) and administered intravenously (i.v.) as a DMSO solution mixed into an aqueous i.v. formulation (see Goetz JP et al., 2005, J. Clin. Oncol. 25:1078-1087, for a description of the administration of 17-allylamino-17- demethoxygeldanamycin.
  • DMSO dimethylsulfoxide
  • i.v. intravenously
  • the compounds may be administered orally in a different formulation.
  • a preferred solvent is DMSO further diluted into a standard aqueous i.v. solution.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension.
  • compositions may be in the form of a lyophilized particulate material, a sterile or aseptically produced solution, a tablet, an ampule, etc.
  • Vehicles such as water (preferably buffered to a physiologically acceptable pH, as for example, in phosphate buffered saline) or an appropriate organic solvent, other inert solid or liquid material such as normal saline or various buffers may be present.
  • the particular vehicle is not critical, and those skilled in the art will know which vehicle to use for any particular utility described herein.
  • a pharmaceutical composition is prepared by mixing, dissolving, binding or otherwise combining the polymer or polymeric conjugate of this invention with one or more water-insoluble or water-soluble aqueous or non-aqueous vehicles. It is imperative that the vehicle, carrier or excipient, as well as the conditions for formulating the composition are such that do not adversely affect the biological or pharmaceutical activity of the active compound.
  • vehicle, carrier or excipient, as well as the conditions for formulating the composition are such that do not adversely affect the biological or pharmaceutical activity of the active compound.
  • Those skilled in the art will know how to formulate and administer the pharmaceutical compositions and carry out the treatment methods disclosed herein. See, for example, Gennaro, AE., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 2003 or latest edition); Pharmaceutical Analysis, Watson D ed. 1 st Edition 1999: Harcourt Pub Limited London, UK.
  • the preferred animal subject of the present invention is a mammal.
  • the invention is particularly useful in the treatment of human subjects.
  • treating is intended the administering to subjects of a pharmaceutical composition comprising a compound as described herein, preferably a benzoquinone.
  • Treating includes administering the agent to subjects at risk for developing a Met-positive tumor prior to evidence of clinical disease, as well as subjects diagnosed with such tumors or cancer, who have not yet been treated or who have been treated by other means, e.g., surgery, conventional chemotherapy, and in whom tumor burden has been reduced even to the level of not being detectable.
  • compositions of the present invention wherein the compound is combined with pharmaceutically acceptable excipient or carrier, may be administered by any means that achieve their intended purpose. Amounts and regimens for the administration of can be determined readily by those with ordinary skill in the clinical art of treating any of the particular diseases. Preferred amounts are described below.
  • the active compounds of the invention may be administered orally, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • the present methods include administration by parenteral routes, including injection or infusion using any known and appropriate route for the subject's disease and condition.
  • Parenteral routes include subcutaneous (s.c.) intravenous (i.v.), intramuscular, intraperitoneal, intrathecal, intracisternal transdermal, topical, rectal or inhalational.
  • i.v. subcutaneous intravenous
  • intramuscular intraperitoneal
  • intrathecal intratracisternal transdermal
  • topical, rectal or inhalational also included is direct intratumoral injection.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the active compound of the invention is administered in a dosage unit formulation containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • an effective amount of the compound is applied directly to the site of surgical removal of a tumor (whether primary or metastatic). This can be done by injection or "topical" application in an open surgical site or by injection after closure.
  • a specified amount of the compound preferably about 1 ng-1 mg, is added to about 700 ml of human plasma that is diluted 1 : 1 with heparinized saline solution at room temperature.
  • Human IgG in a concentration of 500 ⁇ g/dl (in the 700 ml total volume) may also be used.
  • the solutions are allowed to stand for about 1 hour at room temperature.
  • the solution container may then be attached directly to an iv infusion line and administered to the subject at a preferred rate of about 20 ml/min.
  • the pharmaceutical composition is directly infused i.v. into a subject.
  • the appropriate amount preferably about 10 mg - 1 gram , is added to about 250 ml of heparinized saline solution and infused iv into patients at a rate of about 20 ml/min.
  • the composition can be given one time but generally is administered six to twelve times (or even more, as is within the skill of the art to determine empirically).
  • the treatments can be performed daily but are generally carried out every two to three days or as infrequently as once a week, depending on the beneficial and any toxic effects observed in the subject. If by the oral route, the pharmaceutical composition, preferably in a convenient tablet or capsule form, may be administered once or more daily.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration, and all three types of formulation may be used simultaneously to achieve systemic administration of the active ingredient.
  • aerosolized solutions are used.
  • the active protein or small molecule agent may be in combination with a solid or liquid inert carrier material. This may also be packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant.
  • the aerosol preparations can contain solvents, buffers, surfactants, and antioxidants in addition to the protein of the invention.
  • the appearance of tumors in sheaths (“theca”) encasing an organ often results in production and accumulation of large volumes of fluid in the organ's sheath. Examples include (1) pleural effusion due to fluid in the pleural sheath surrounding the lung, (2) ascites originating from fluid accumulating in the peritoneal membrane and (3) cerebral edema due to metastatic carcinomatosis of the meninges. Such effusions and fluid accumulations generally develop at an advanced stage of the disease.
  • the present invention contemplates administration of the pharmaceutical composition directly administration into cavities or spaces, e.g. , peritoneum, thecal space, pericardial and pleural space containing tumor.
  • the agent is directly administered into a fluid space containing tumor cells or adjacent to membranes such as pleural, peritoneal, pericardial and thecal spaces containing tumor. These sites display malignant ascites, pleural and pericardial effusions or meningeal carcinomatosis .
  • the drug is preferably administered after partial or complete drainage of the fluid (e.g., ascites, pleural or pericardial effusion ) but it may also be administered directly into the undrained space containing the effusion, ascites and/or carcinomatosus.
  • the compound's dose may vary from 1 nanogram to 1 mg, preferably, 10 ng to 100 ⁇ g, and given every 3 to 10 days. It is continued until there is no reaccumulation of the ascites or effusion. Therapeutic responses are considered to be no further accumulation of four weeks after the last intrapleural administration.
  • the active compound may be incorporated into topically applied vehicles such as salves or ointments, as a means for administering the active ingredient directly to the affected area. Scarification methods, known from studies of vaccination, can also be used.
  • the carrier for the active agent may be either in sprayable or nonsprayable form. Non-sprayable forms can be semi-solid or solid forms comprising a carrier indigenous to topical application and having a dynamic viscosity preferably greater than that of water. Suitable formulations include, but are not limited to, solution, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like.
  • preferred vehicles for non-sprayable topical preparations include ointment bases, e.g., polyethylene glycol- 1000 (PEG-1000); conventional creams such as HEB cream; gels; as well as petroleum jelly and the like.
  • compositions are liposomes or other timed-release or gradual release carrier or drug delivery device known in the art
  • Anti-cancer chemotherapeutic drugs useful in this invention include but are not limited to antimetabolites, anthracycline, vinca alkaloid, anti-tubulin drugs, antibiotics and alkylating agents.
  • Representative specific drugs that can be used alone or in combination include cisplatin (CDDP), adriamycin, dactinomycin, mitomycin, carminomycin, daunomycin, doxorubicin, tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), verapamil, podophyllotoxin, 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, camptothecin, actinomycin-D, mitomycin C, aminopterin, combretastatin(s) and derivatives and prodrugs thereof.
  • CDDP cisplatin
  • adriamycin adriamycin
  • dactinomycin mitomycin
  • carminomycin daunomycin
  • doxorubicin doxorubicin
  • any one or more of such drugs, newer drugs targeting oncogene signal transduction pathways, or that induce apoptosis or inhibit angiogenesis, and biological products such as nucleic acid molecules, vectors, antisense constructs, siRNA constructs, and ribozymes, as appropriate, may be used in conjunction with the present compounds and methods.
  • agents and therapies include, radiotherapeutic agents, antitumor antibodies with attached anti-tumor drugs such as plant-, fungus-, or bacteria- derived toxin or coagulant, ricin A chain, deglycosylated ricin A chain, ribosome inactivating proteins, sarcins, gelonin, aspergillin, restricticin, a ribonuclease, a epipodophyllotoxin, diphtheria toxin, or Pseudomonas exotoxin.
  • radiotherapeutic agents such as plant-, fungus-, or bacteria- derived toxin or coagulant
  • ricin A chain deglycosylated ricin A chain
  • ribosome inactivating proteins such asarcins, gelonin, aspergillin, restricticin, a ribonuclease, a epipodophyllotoxin, diphtheria toxin, or Pseudomonas ex
  • Additional cytotoxic, cytostatic or anti-cellular agents capable of killing or suppressing the growth or division of tumor cells include anti-angiogenic agents, apoptosis-inducing agents, coagulants, prodrugs or tumor targeted forms, tyrosine kinase inhibitors, antisense strategies, RNA aptamers, siRNA and ribozymes against VEGF or VEGF receptors. Any of a number of tyrosine kinase inhibitors are useful when administered together with, or after, the present compounds. These include, for example, the 4-aminopyrrolo[2,3-d]pyrimidines (U.S. Pat. No. 5,639,757).
  • small organic molecules capable of modulating tyrosine kinase signal transduction via the VEGF -R2 receptor are the quinazoline compounds and compositions (U.S. Pat. No. 5,792,771).
  • Other agents which may be employed in combination with the present invention are steroids such as the angiostatic 4,9(1 l)-steroids and C 21 -oxygenated steroids (U.S. Pat. No. 5,972,922).
  • Thalidomide and related compounds, precursors, analogs, metabolites and hydrolysis products may also be used in combination to inhibit angiogenesis. These thalidomide and related compounds can be administered orally.
  • Other anti-angiogenic agents that cause tumor regression include the bacterial polysaccharide CMlOl (currently in clinical trials) and the antibody LM609. CMlOl induces neovascular inflammation in tumors and downregulates expression VEGF and its receptors.
  • Thrombospondin (TSP-I) and platelet factor 4 (PF4) are angiogenesis inhibitors that associate with heparin and are found in platelet ⁇ granules.
  • Interferons and matrix metalloproteinase inhibitors are two other classes of naturally occurring angiogenic inhibitors that can be used.
  • Tissue inhibitors of metalloproteinases are a family of naturally occurring MMPFs that also inhibit angiogenesis.
  • Other well- studied anti-angiogenic agents are angiostatin, endostatin, vasculostatin, canstatin and maspin.
  • Chemotherapeutic agents are administered as single agents or multidrug combinations, in full or reduced dosage per treatment cycle.
  • the combined use of the present compositions with low dose, single agent chemotherapeutic drugs is particularly preferred.
  • the choice of chemotherapeutic drug in such combinations is determined by the nature of the underlying malignancy.
  • cisplatin is preferred.
  • a microtubule inhibitor such as taxotere is the preferred.
  • 5-FU is preferred.
  • Low dose as used with a chemotherapeutic drug refers to the dose of single agents that is 10-95% below that of the approved dosage for that agent (by the U.S. Food and Drug Administration, FDA).
  • each drug dose is reduced by the same percentage. A reduction of >50% of the FDA approved dosage is preferred although therapeutic effects are seen with dosages above or below this level, with minimal side effects.
  • Multiple tumors at different sites may be treated by systemic or by intrathecal or intratumoral administration of the benzoquinone and other compounds described herein.
  • the present compounds may be tested for therapeutic efficacy in well established rodent models which are considered to be representative of a human tumor.
  • the overall approach is described in detail in Geran, R.I. et ah, "Protocols for Screening Chemical Agents and Natural Products against Animal Tumors and Other Biological Systems (3d Ed)", Cane. Chemother. Reports, Part 3, 5:1-112; and Plowman, J et ah, In: Teicher, B, ed., Anticancer Drug Development Guide: Preclinical Screening, Clinical Trials and Approval, Part II: In Vivo Methods, Chapter 6, "Human Tumor Xenograft Models in NCI Drug Development," Humana Press Inc., Totowa, NJ, 1997.
  • Xenografts are generally not utilized for drug evaluation until the volume-doubling time has stabilized, usually around the fourth or fifth passage.
  • the in vivo growth characteristics of the xenografts determine their suitability for use in the evaluation of test agent antitumor activity, particularly when the xenografts are utilized as early stage s.c. models.
  • an early stage s.c. model is defined as one in which tumors are staged to 63-200 mg prior to the initiation of treatment. Growth characteristics considered in rating tumors include take -rate, time to reach 200 mg, doubling time, and susceptibility to spontaneous regression. As can be noted, the faster- growing tumors tend to receive the higher ratings.
  • transgenic mouse models Any of a number of transgenic mouse models known in the art can be used to test the present compounds.
  • a particularly useful murine human HGF/SF transgenic model has been described by one of the present inventors and his colleagues and may be used to test the present compounds against human tumor xenografts in vivo. See, Zhang YW et al. (2005) Oncogene 24:101-106; U.S. Pat.App Ser. No. 60/587,044, which references are incorporated by reference in their entirety. Other longer- known models are described below.
  • Such s. c. -implanted tumor xenograft models are used to evaluate the antitumor activity of test agents under conditions that permit determination of clinically relevant parameters of activity, such as partial and complete regression and duration of remission (Martin DS et al, Cancer Treat Rep (55:37-38 (1984); Martin DS et ah, Cancer Res. 46:2189-2192 (1986); Stolf ⁇ , RL et al., J. Natl Cane Inst 80:52-55 (1988)).
  • Tumor growth is monitored and test agent treatment is initiated when tumors reach a weight range of 100- 400 mg (staging day, median weights approx. 200 mg), although depending on the xenograft, tumors may be staged at larger sizes.
  • Tumor sizes and body weights are obtained approximately 2 times/wk.
  • software programs developed by staff of the Information Technology Branch of DTP of the NCI
  • data are stored, various parameters of effects are calculated, and data are presented in both graphic and tabular formats.
  • Parameters of toxicity and antitumor activity are defined as follows:
  • Toxicity Both drug-related deaths (DRD) and maximum percent relative mean net body weight losses are determined.
  • a treated animal's death is presumed to be treatment-related if the animal dies within 15 d of the last treatment, and either its tumor weight is less than the lethal burden in control mice, or its net body weight loss at death is 20% greater than the mean net weight change of the controls at death or sacrifice.
  • a DRD also may be designated by the investigator.
  • the mean net body weight of each group of mice on each observation day is compared to the mean net body weight on staging day. Any weight loss that occurs is calculated as a percent of the staging day weight.
  • Optimal % T/C Changes in tumor weight (A weights) for each treated (T) and control (C) group are calculated for each day tumors are measured by subtracting the median tumor weight on the day of first treatment (staging day) from the median tumor weight on the specified observation day. These values are used to calculate a percent T/C as follows:
  • % T/C ( ⁇ T/ ⁇ C) ⁇ 100 where ⁇ T>0 or
  • Tumor growth delay This is expressed as a percentage by which the treated group weight is delayed in attaining a specified number of doublings; (from its staging day weight) compared to controls using the formula:
  • T and C are the median times (in days) for treated and control groups, respectively, to attain the specified size (excluding tumor- free mice and DRDs).
  • the growth delay is expressed as percentage of control to take into account the growth rate of the tumor since a growth delay based on (T - C) alone varies in significance with differences in tumor growth rates. 4.
  • Net log cell kill An estimate of the number of logio units of cells killed at the end of treatment is calculated as:
  • tumor smaller than 4 x 4 mm or 5 x 5 mm (32 and 63 mg, respectively) is questionable. Also, once a relatively large tumor has regressed to 63 mg, the composition of the remaining mass may be only fibrous material/scar tissue. Measurement of tumor regrowth following cessation of treatment provides a more reliable indication of whether or not tumor cells survived treatment. Most xenografts that grow s.c. may be used in an advanced-stage model, although for some tumors, the duration of the study may be limited by tumor necrosis. As mentioned previously, this model enables the measurement of clinically relevant parameters and provides a wealth of data on the effects of the test agent on tumor growth.
  • the investigator is ensured that angiogenesis has occurred in the area of the tumor, and staging enables "no-takes" to be eliminated from the experiment.
  • the model can be costly in terms of time and mice.
  • the passage time required before sufficient mice can be implanted with tumors may be at least ⁇ 4 wks, and an additional 2-3 wks may be required before the tumors can be staged.
  • more mice as many as 50-100% more than are needed for actual drug testing must be implanted.
  • the "early treatment model” is defined as one in which treatment is initiated before tumors are measurable, i.e., ⁇ 63 mg.
  • the "early stage” model as one in which treatment is initiated when tumor size ranges from 63-200 mg. The 63-mg size is used because it indicates that the original implant, about 30 mg, has demonstrated some growth. Parameters of toxicity are the same as those for the advanced-stage model; parameters of antitumor activity are similar.
  • %T/C values are calculated directly from the median tumor weights on each observation day instead of being measured as changes ( ⁇ ) in tumor weights, and growth delays are based on the days after implant required for the tumors to reach a specified size, e.g., 500 or 1000 mg.
  • Tumor-free mice are recorded, but may be designated as "no-takes” or spontaneous regressions if the vehicle-treated control group contains >10% mice with similar growth characteristics.
  • a "no-take” is a tumor that fails to become established and grow progressively.
  • a spontaneous regression (graft failure) is a tumor that, after a period of growth, decreases to ⁇ 50% of its maximum size. Tumor regressions are not normally recorded, since they are not always a good indicator of antineoplastic effects in the early stage model.
  • a major advantage of the early treatment model is the ability to use all implanted mice, which is why a good tumor take-rate is required. In practice, the tumors most suitable for this model tend to be the faster-growing ones. Challenge Survival
  • titration groups are included to establish a tumor doubling time for use in logio cell kill calculations.
  • a death (or sacrifice) may be designated as drug-related based on visual observations and/or the results of necropsy. Otherwise, treated animal deaths are-designated as treatment-related if the day of death precedes the mean day of death of the controls (-2SD) or if the animal dies without evidence of tumor within 15 days of the last treatment.
  • the test agent is evaluated following i.p. administration at multiple dose levels.
  • the activity ratings are based on the optimal effects attained with the maximally tolerated dose ( ⁇ LD 2 o) of each drug for a given treatment schedule which is selected on the basis of the doubling time of a given tumor, with longer intervals between treatments for slower growing tumors.
  • ⁇ LD 2 o maximally tolerated dose
  • ⁇ 40 were produced in the melanoma group by at least 2, and as many as 10, clinical drugs.
  • the number of responses appeared to be independent of doubling time and histological type with a range in the number of responses observed for tumors (seen in each subpanel of other tumor types as well).
  • these tumors models (across all tumors) were quite refractory to standard drug therapy; the tumors did not respond to any of the drugs tested in 30 of 48 (62.5%) of all tumors.
  • the in vitro primary screens provide a basis for selecting the most appropriate tumor lines to use for follow-up in vivo testing, with each compound tested only against xenografts derived from cell lines demonstrating the greatest sensitivity to the agent in vitro.
  • the early strategy for in vivo testing emphasized the treatment of animals bearing advanced-stage tumors. Based on the specific information available to guide dose selection here, much lower doeses than those used for typical test agents are selected.
  • Single mice are preferably treated with single ip bolus doses of between 1 pg/kg and and 1 mg/kg and observed for 14 d. Sequential 3-dose studies may be conducted as necessary until a nonlethal dose range is established. The test agent is then evaluated preferably in three s.c.
  • xenograft models using tumors that are among the most sensitive to the test agent in vitro and that are suitable for use as early stage models.
  • the compounds are administered ip, as suspensions if necessary, on schedules based, with some exceptions, on the mass doubling time of the tumor. For example, for doubling times of 1.3-2.5, 2.6-5.9, and 6-10 d, preferred schedules are: daily for five treatments (qd x 5), every fourth day for three treatments (q4d x 3), and every seventh day for three treatments (q7d x 3). For most tumors, the interval between individual treatments approximates the doubling time of the tumors, and the treatment period allows a 0.5-1.0 logio unit of control tumor growth.
  • the tumor sizes of the controls at the end of treatment should range from 500-2000 mg, which allows sufficient time after treatment to evaluate the effects of the test agent before it becomes necessary to sacrifice mice owing to tumor size.
  • Detailed Drug Studies Once a compound has been identified as demonstrating in vivo efficacy in initial evaluations, more detailed studies are designed and conducted in human tumor xenograft models to explore further the compound's therapeutic potential. By varying the concentration and exposure time of the tumor cells and the host to the drug, it is possible to devise and recommend treatment strategies designed to optimize antitumor activity. The importance of "concentration x time" on the antitumor effects of test agents were well illustrated by data obtained with amino-20M-camptothecin (Plowman, J. et ah, 1997 ', supra). Those results indicated that maintaining the plasma concentration above a threshold level for a prolonged period of time was required for optimal therapeutic effects.
  • Late metastasis involves the steps of attachment and extravasation of tumor cells, local invasion, seeding, proliferation and angiogenesis.
  • a reporter gene preferably the green fluorescent protein (GFP) gene
  • Cells are injected, preferably iv, and metastases identified after about 14 days, particularly in the lungs but also in regional lymph nodes, femurs and brain. This mimics the organ tropism of naturally occurring metastases in human melanoma.
  • GFP-expressing melanoma cells (10 6 cells per mouse) are injected i.v. into the tail veins of nude mice. Animals are treated with a test composition at lOO ⁇ g/animal/day given q.d. IP.
  • Single metastatic cells and foci are visualized and quantitated by fluorescence microscopy or light microscopic histochemistry or by grinding the tissue and quantitative colorimetric assay of the detectable label.
  • Representative mice are subjected to histopatho logical and immunocytochemical studies to further document the presence of metastases throughout the major organs. Number and size (greatest diameter) of the colonies can be tabulated by digital image analysis, e.g., as described by Fu, Y. S. et al., Anat. Quant. Cytol. Histol. 77:187-195 (1989)).
  • explants of lung, liver, spleen, para-aortic lymph nodes, kidney, adrenal glands and s.c. tissues are washed, minced into pieces of 1-2 mm 3 and the pieces pulverized in a Tekman tissue pounder for 5 min.
  • the pulverized contents are filtered through a sieve, incubated in a dissociation medium (MEM supplemented with 10% FCS, 200 U/ml of collagenase type I and 100 ⁇ g/ml of DNase type I) for 8 hr at 37 0 C with gentle agitation.
  • a dissociation medium MEM supplemented with 10% FCS, 200 U/ml of collagenase type I and 100 ⁇ g/ml of DNase type I
  • the resulting cell suspension is washed and resuspended in regular medium ⁇ e.g., MEM with 10% FCS supplemented with the selecting antibiotic (G-418 or hygromycin).
  • regular medium ⁇ e.g., MEM with 10% FCS supplemented with the selecting antibiotic (G-418 or hygromycin).
  • the explants are fed and the number of clonal outgrowths of tumor cells is determined after fixation with ethanol and staining with an apprpriate ligand such as a monoclonal antibody to a tumor cell marker. The number of colonies is counted over an 80-cm 2 area.
  • clonal outgrowths are not fixed and stained but rather are retrieved fresh with cloning rings and pooled after only a few divisions for other measurements such as secretion of collagenases (by substrate gel electrophoresis) and Matrigel invasion.
  • Matrigel invasion assays are described herein, though it is possible to use assays described by others (Hendrix, M.J.C. et al, Cancer Lett., 38: 137-147 (1987); Albini, A. et al, Cancer Res., 47 3239-3245 (1987); Melchiori, A., Cancer Res. 52:2353-2356 (1992)).
  • mice All experiments are performed with groups that preferably have 10 mice. Results are analyzed with standard statistical tests. Depending on the tumor, i.v. injections of 0.2-10 x 10 5 tumor cells 1 week after an s.c. flank injection of an equal number of tumor cells followed by an additional 5-week interval yielded a ratio of hematogenous: spontaneous pulmonary metastases and an overall pulmonary tumor burden that is convenient for evaluation.
  • the model may peroit retrieval of numerous extrapulmonary metastatic clones from spleen, liver, kidneys, adrenal gland, para-aortic lymph nodes and s.c. sites, most of which likely represent spontaneous metastases from the locally growing tumor.
  • Doses of a composition under test are determined as using, inter alia, appropriate animal models of the tumor of cancer of interest.
  • a pharmaceutical composition of the present invention is administered.
  • a treatment consists of injecting the subject with, for example, 1, 100 and 1000 ng of the compound intravenously in 200 ml of normal saline over a one-hour period. Treatments are given 3x/week for a total of 12 treatments. Patients with stable or regressing disease are treated beyond the 12th treatment. Treatment is given on either an outpatient or inpatient basis as needed.
  • Tumor response criteria are those established by the International Union against Cancer and are listed below.
  • the efficacy of the therapy in a patient population is evaluated using conventional statistical methods, including, for example, the Chi Square test or Fisher's exact test. Long-term changes in and short term changes in measurements can be evaluated separately.
  • a GA derivative compound particularly a benzoquinone thereof, to be useful in accordance with this invention, it should demonstrate activity at the nanomolar level in at least one of the in vitro, biochemical, or molecular assays described herein and also have potent antitumor activity in vivo.
  • EXAMPLE I Materials and Methods Cell lines and drugs.
  • MDCK canine kidney epithelial cells
  • DBTRG human glioblastoma multiforme cells
  • Both cell lines were grown in Dulbecco's modified Eagle's Medium (DMEM' GibcoTM, Invitrogen Corp.) and supplemented with 10% fetal bovine serum (FBS; from Hyclone) as previously described (Xie et al., supra).
  • C 6 -GA and TEG-GA coupled gel-affinity beads (Fig. 4/5), as well as C 6 5- acetomido-2-methoxy-3-methylbenzoquinone (quinone) were prepared as previously described (Shen et al., supra). Briefly, quinone or GA (1.5 equivalents to one affinity gel bead equivalent) were stirred with the diamine linker 1 ,6-diaminohexane or tetraethylene glycol diamine (TEG) at 5-10 equivalents of TEG to one equivalent of quinone or GA in chloroform at room temperature. Upon the complete conversion of the starting material (monitored by thin layer chromatography), the mixture was washed sequentially with diluted aqueous NaOH and brine.
  • quinone or GA 1.5 equivalents to one affinity gel bead equivalent
  • TEG tetraethylene glycol diamine
  • Control beads were made by conjugating the affinity gel bead material with a short -chain analogue that has no affinity for HSP90.
  • Affi-Gel 10 beads Bio-Rad
  • JV-(6-aminohexyl) acetamide M. Zoratti et ah, Biochim Biophys Acta 1241, 139 (1995)
  • the GA-conjugated and control affinity beads were washed in five volumes of TNESV buffer (50 mM Tris-HCl, pH 7.5, 20 mM Na 2 MoO 4 , 1% Triton X-IOO, 150 mM NaCl, and 1 mM Na 3 VO 4 ) three times and rotated overnight in TNESV at 4 0 C to hydrolyze any unreacted iV-hydroxysuccinimide, then rocked in 1% BSA in TNESV (1 :10) at room temperature for at least 3 h. After washing with TNESV three additional times, beads were resuspended in 50% TNESV and stored at -8O 0 C.
  • TNESV buffer 50 mM Tris-HCl, pH 7.5, 20 mM Na 2 MoO 4 , 1% Triton X-IOO, 150 mM NaCl, and 1 mM Na 3 VO 4
  • PVDF polyvinylidene difluoride
  • Excised gel bands were digested in-gel with trypsin in a ProGest® robot (Genomic Solutions) following the procedure described by Shevchenko et al. (Anal Chem 68, 850 (1996)). Extracted peptides were analyzed on a Waters/Micromass Q-Tof API mass spectrometer equipped with a Waters/Micromass CapLC HPLC. Sample was loaded onto a Michrom Cap Trap concentrating/desalting trap column installed on a 10-port switching valve (Valco-Vici) at a 30 ⁇ l/min flow rate.
  • valve was switched to allow nanoflow reverse phase gradient elution using 0.1% formic acid in water for buffer A and 0.1% formic acid in 90% acetonitrile for buffer B.
  • Gradient elution at an approximate 250 nl/min flow rate was achieved via flow- splitting through a stainless-steel cross (Valco-Vici).
  • Analytical separation of peptides was performed on a 75 ⁇ m ID x 15 cm PicoFrit® column (New Objectives) packed in-house (Michrom C- 18 Magic® 5 ⁇ m particles). The Q-Tof was operated in DDA mode for automated MS to MS/MS switching.
  • the DNA encoding amino acids 1-283 of human VDAC was isolated from human ovary cDNA (Human Ovary Quick-clone cDNA, Clonetech) by PCR using as primers:
  • the rhVDAC-l-pET-21a construct was transformed into BL21(DE3) and expression of the His-tagged rh VD AC-I was induced by 1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) for 4hr at 37°C with vigorous shaking. Protein purification was based upon a published method (Y. Shi et al., Biochem Biophys Res Commun 303, 475 (2003)).
  • the biomass was suspended in buffer A (20 mM Tris, pH 7.9, 500 mM NaCl, and 1 mM PMSF) and homogenized by pulse sonication on ice.
  • the inclusion bodies were incubated in 10 ml binding buffer with 1 mg/ml lysozyme for 15 min and homogenized by sonication once again. After centrifugation at 7000 rpm for 15 min, pellet was solubilized with 6 M guanidine-HCl in buffer A for 3 h with gentle stirring, followed by adjusting guanidine-HCl to 4M for another 1 hour with gentle stirring at 4°C.
  • the supernatant containing the denatured rh VD AC-I was collected by centrifugation at 18,000 ⁇ g for 20 min.
  • Two ml of Nico beads (Novagene) pre-equilibrated with buffer B (4M guanidine-HCl, 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, and 10% glycerol) were loaded with 10 ml denatured protein supernatant, followed by washes with 5 volumes of buffer B.
  • Mitochondrial Isolation and GA-Coupled Bead Precipitation Mitochondria were isolated from cell lysates by using a commercial isolation kit
  • the washed mitochondrial pellet was lysed with TNESV supplemented with 1% Triton X-100 and CompleteTM proteinase inhibitors (Roche Molecular Biochemicals). GA-conjugated affinity bead pull-down assays followed by immunoblotting analysis with antibodies directed against HSP90 and VDAC were performed as described above.
  • MDCK total cell lysate (equivalent to 500 ⁇ g total protein for each sample) was first adjusted to 500 ⁇ l with TNSEV buffer. Twenty ⁇ l of GA-conjugated beads were added to each sample, and the suspensions were rotated at 4 0 C for 4hr. Beads were collected by centrifugation at
  • HGF/SF 60 units/ml was added to all wells (with the exception of wells used to calculate basal growth and plasmin activation). Twenty- four hrs after drug/HGF/SF addition, plates were processed for the determination of plasmin activity as follows.
  • MDCK cells were cultured on 5 x 5-mm plastic coverslips and were transferred to a microperfusion chamber on the stage of an inverted microscope.
  • Whole-cell voltage clamp was accomplished with an Axopatch 200B (Axon Instr. Co., Union City, CA) using suction-ruptured patches primarily from single cells or from cells on the edge of islands comprising 4 or 5 cells.
  • Borosilicate glass capillaries (1.2 mm OD, 0.68 ID, type EN-I, Garner Glass Co., Claremont, CA) were used to fabricate patch pipettes (3-8 M ⁇ in the bath solution) with a Brown-Flaming horizontal micropipette puller (P-87, Sutter Instruments, San Rafael, CA).
  • the internal (pipette) solution for measuring ion currents contained (mM): 140 K gluconate or Cs aspartate, 2 MgCl 2 , 1 CaCl 2 , 10 EGTA, 10 HEPES, pH 7.2 with KOH.
  • the external solution was exchanged for one in which cations were substituted with an impermeable organic cation (in mM): 156 JV-methyl-D-glucamine (NMDG), 1 MgCl 2 , 10 HEPES, 10 glucose, pH 7.4 with 1 N HCl.
  • Electrophysiological recordings were procured and analyzed using PClamp8® software (Axon Instr.
  • MDCK cells and human DBTRG glioblastoma cells which are sensitive to certain GA drugs when measured in uPA-plasmin activation assays and in vitro scattering and invasion assays .
  • HSP90 was mainly in the post-nuclear pull-down supernatant whereas only VDAC was detected in the post-mitochondrial GA pull-down supernatant (Fig. 1, lanes 4-6).
  • VDAC is a component of the mitochondrial PTP, a calcium-, voltage- sensitive, and pH-sensitive pore that opens when calcium levels increase, especially when accompanied by oxidative stress (Green et ah, supra; Marzo et ah, supra).
  • a number of well- known inhibitors of the PTP and VDAC including 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid, disodium salt (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid, disodium salt (H 2 DIDS) (D.
  • DIDS is a nonspecific, voltage-dependent VDAC inhibitor that prevents O 2 diffusion from the intermembrane space to the cytoplasm. At 0.5 mM, DIDS inhibited superoxide production in isolated heart mitochondria (Han et al, supra). Both DIDS (not shown) and H 2 DIDS at 100 ⁇ M inhibited uPA-plasmin activation (Fig. 2). Importantly, both UbO and Decyl-Ub benzoquinones, both of which are believed to bind to VDAC (E.
  • CsA Cyclosporin A
  • Benzoquinone moieties of GA derivatives shown below were tested for activity in the MDCK-HGF/SF induced uPA assay.
  • One derivative, 5-Acetomido-2-(l-aziridinyl)-3-methyl- benzoquinone (ARQ) the benzoquinone found in 17-(l-aziridinyl)-17- demethoxygeldanamycin (ARG) displayed nanomolar inhibitory activity (Table 2 and Fig. 3) and, therefore, according to the present invention, has therapeutic activity, based on its inhibitory concentration above, which is two orders of magnitude lower than the inhibitory concentration of UbO and in the same potency range as several of the "active" GA derivatives (the full sized molecules).
  • Table 2B A summary of the concentration ranges in which various of the GA and derivative compounds and the benzoquinones of the present invention exert their inhibitory action in the uPA assay described herein are shown in Table 2B
  • UbO, Decyl-Ub, and GA each include a benzoquinone group and are members of a family of "anti-tumor quinones" that are metabolized by NADPH: quinone oxidoreductase 1 (NQOl, DT-diaphorase) (D. Ross, Drug Metab Rev 36, 639 (2004)) and are processed in mitochondria.
  • NADPH quinone oxidoreductase 1
  • NQOl, DT-diaphorase quinone oxidoreductase 1
  • UbO and Decyl-Ub blocked PTP pore activity by regulating Ca 2+ permeability, an effect that could be reversed by increasing Ca 2+ concentration (Martinucci et al, supra).
  • HGF/SF stimulation of cell migration increases Ca 2+ influx through a plasma-membrane transient receptor potential cation channel, TRPVl (J.
  • HGF/SF 100 ng/ml was added where indicated. Inward current increased within minutes after adding HGF/SF, and this increase reversed immediately when N-methyl-D-glucamine (NMDG) was substituted for all external cations (Fig. 4B). Both inward and outward current increased further over an additional ten minutes.
  • the overall time course is similar to the time course of epidermal growth factor (EGF) stimulation of vesicular translocation and insertion of TRP channels and their corresponding increases in [Ca 2+ J 1 concentration (VJ Bezzerides et al., Nat Cell Biol 6, 709 (2004)).
  • EGF epidermal growth factor
  • uPA-plasmin inhibition index is the negative loglO of the drug concentration at which 50% inhibition of uPA occurs (Webb et. al. 2000).
  • GA therefore is a highly active inhibitor of Ca 2+ flux.
  • Membrane currents plotted as a function of the voltage ramps were taken at the indicated times.
  • HGF/SF increased the slope conductance accompanied by a shift in reversal potential toward 0 mV, whereas subsequent addition of GA (10 ⁇ 12 M) reduced the slope conductance (Table 2).
  • benzoquinone drugs all reduce HGF/SF-induced cation current indicates that they have a common target.
  • the fact that the GA displays activity at picomolar levels is significant.
  • a negative control compound, radicicol was tested; this compound (2, 3) has much higher affinity for HSP90 than GA and has activity against uPA at nanomolar concentrations, presumably where it inhibits HSP90 function.
  • HGF/SF stimulates a nonselective cation current in MDCK cells, similar to its effect on HepG2 cells (Vriens et al., supra). It is significant that UbO and Decyl-Ub, known inhibitors of the mitochondrial VDAC and PTP (Fontaine et al., supra), also reduce HGF/SF- activated increases in slope conductance in the same concentration range that inhibited uPA- plasmin activity (cf. Fig. 3 to Fig.
  • nS nanoSiemens
  • pF picoFarads
  • GA targets HSP90, and, because of HSP90's function as a chaperone, has a broad spectrum of effects, including on Met (Webb et al., supra; Xie et al, supra). Because of this, effects mediated through mitochondria would be masked. Inhibition of cationic currents in "control" cells treated with GA at picomolar levels is shown in Table 2). UbO also has inhibitory activity in control cells not treated with HGF/SF (Table 3). This is further evidence that benzoquinone compounds act as general inhibitors of mitochondrial activity, and can inhibit cell invasion activity at lower concentrations than those affecting HSP90.
  • UbO also has inhibitory activity in control cells not treated with HGF/SF (Table 1), further supporting the notion that the benzoquinone compounds are general inhibitors of mitochondrial activity that is associated with cell invasion.
  • GA, UbO, and Decyl-Ub each including benzoquinone, inhibit both HGF/SF-induced membrane current and uPA activity.
  • GA acts at nanomolar concentrations whereas both UbO and Decyl-Ub act at micromolar concentrations (Fig. 3, Table 3).
  • the benzoquinone compound, ARQ acts at nanomolar levels. This is strong evidence that GA has quinone-like bioactivity, as previously indicated (S.
  • Redox-cycling quinones can participate in catalytic electron transfer processes via an intermediate one-electron reduced semiquinone form and transition to either the quinone (fully oxidized) or the hydroquinone (fully reduced) state.
  • UbO and Decyl-Ub both tested by the present inventors, belong to this category.
  • GA works at concentrations orders of magnitude lower than UbO and Decyl-Ub, both in inhibiting cell scattering and uPA- plasmin activation as well as in whole-cell voltage clamp assays (Table3).

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Abstract

Derivatives of geldanamycin (GA), a benzoquinone ansamycin antibiotic, are in clinical trials as anticancer drugs. While, at nanomolar concentrations, these drugs are known to target HSP90 chaperone activity, second anticancer activity that inhibits HGF/SF-mediated tumor cell invasion at nanomolar concentrations has been found. Disclosed is the identification of the outer membrane mitochondrial voltage-dependent anion channel (VDAC) protein, a component of the permeability transition pore (PTP), as a novel GA binding protein and a demonstration that GA inhibits membrane potential at picomolar levels. Uubiquinone 0 and decyl-ubiquinone, known benzoquinone inhibitors of mitochondrial PTP function like GA, inhibit HGF/SF induced urokinase (uPA) plasmin protease activation and HGF/SF activated membrane currents in whole-cell voltage clamp assays, with GA blocking at picomolar levels. The results disclosed regarding mechanism of action are a major step in harnessing such drugs for use for treating highly invasive cancers like glioblastoma.

Description

GELDANAMYCINS AND THEIR QUINONE MOIETIES INHIBIT CANCER BY ACTING ON MITOCHONDRIAL VOLTAGE-DEPENDENT ANION CHANNEL
(VDAC) PROTEIN
Field of the Invention The present invention in the field of cancer pharmacology is directed to quinone and hydroquinone chemical moieties present within geldanamycin and its derivatives), that inhibit cancer cell activities at nanomolar concentrations and act at the outer membrane mitochondrial voltage-dependent anion channel (VDAC) protein. These compounds are used to inhibit HGF-dependent, Met-mediated tumor cell activation, growth, invasion, and metastasis and serve as potent anticancer agents.
DESCRIPTION OF THE BACKGROUND ART
Geldanamycin (GA) is a benzoquinone ansamycin antibiotic. GA derivatives are in clinical trials as anticancer drugs. L. Whitesell et al. (Proc Natl Acad Sci USA91, 8324 (1994)) discovered that the molecular chaperone, HSP90, is a target for GA. GA binds to the HSP90 binding pocket, blocking ATP hydrolysis which is required for HSP90 chaperone function. GA-induced inactivation of HSP90 function results in the degradation of a large set of oncoproteins, thereby accounting for its therapeutic action. These drugs target HSP90 chaperone activity at nanomolar concentrations. T he present inventors and their colleagues (CP Webb et al., Cancer Res 60, 342 (2000); Q. Xie et al., Oncogene 24, 3697 (2005); Y. Shen et al, Bioorg Med Chem 13, 4960 (2005)) discovered an anticancer activity that inhibits HGF/SF-mediated tumor cell invasion at lower concentrations without affecting HSP90. See also, PCT Publication WO 2005/095437, by Xie et al. (disclosing an invention by the present inventors and colleagues) which is incorporated by reference in its entirety. The outer membrane mitochondrial voltage-dependent anion channel (VDAC) protein is a component of the mitochondrial permeability transition pore (PTP), a calcium-, voltage-, and pH-sensitive pore that opens when calcium levels increase, especially when accompanied by oxidative stress (DR Green et al., Science 281, 1309 (1998). I. Marzo et al., J Exp Med 187, 1261 (1998)). As disclosed herein the present invention is directed to the targeting of VDAC by GA and derivatives, with an emphasis on this activity mediated by redox-cycling benzoquinone/ semiquinone/ hydroquinone moieties of GA or its derivatives. Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
SUMMARY OF THE INVENTION
The present invention identifies VDAC as a novel GA binding protein and shows that GA inhibits membrane current at pM levels. Moreover, ubiquinone 0 and decyl- ubiquinone, known benzoquinone inhibitors of mitochondrial PTP function that act like GA, also inhibit HGF/SF induced urokinase (uPA) plasmin protease activation as well as action currents measured in whole-cell voltage clamp assays with GA blocking at pM levels. Disclosed herein is the discovery that a benzoquinone moiety of a novel aziridinyl derivative of GA has nanomolar inhibitory activity against HGF/SF induced uPA activity identifying this compound as a useful agent against cancer. The present invention is directed to a redox-cycling benzoquinone, semiquinone or hydroquinone compound, or a pharmaceutically acceptable salt of the benzoquinone , which compound has the property of inhibiting (a) the activation of Met by HGF/SF in cancer cells and (b) HGF/SF-induced tumor cell invasion, which benzoquinone, semiquinone, hydroquinone or compound or salt binds to and inhibits the activity of mitochondrial VDAC protein of cells.
The above benzoquinone is preferably one that is present in the structure of GA or a 17-alkylamino 17-demethoxy derivative of GA.
The above compound inhibits VDAC to an extent that is equal to or exceeds the inhibitory action of mitochondrial specific VDAC inhibitors ubiquinone (UbO) or decyl- ubiquinone (Decyl-Ub).
The above compound preferably inhibits non-selective cation currents in the cells at subnanomolar concentrations, including at concentrations below <10"10M.
The above compound preferably inhibits Ca2+ influx, thereby acting as a calcium channel blocker. The compound is preferably selected from the group consisting of:
5 - Acetamido-2-methoxy-3 -methylbenzoquinone (GQ), 5 - Acetamido-2-(2-fluoroethyl)amino-3 -methylbenzoquinone (FEQ),
5 -Acetamido-2-allylamino-3 -methylbenzoquinone (AAQ), 5 - Acetamido-2-( 1 -aziridinyl)-3 -methylbenzoquinone (ARQ), 5-Acetamido-2-(2-acetamidoethyl)amino-3-methylbenzoquinone (AAEQ), 5 -Acetamido-2-(6-acetamidohexyl)amino-3 -methylbenzoquinone (AAHQ), 5 - Acetamido-2-[2- [2-(2-acetamidoethyloxy)ethoxy] ethyl] amino-3 -methylbenzoquinone (AATEQ), and
5 -Acetamido-2-carboxymethylamino-3 -methylbenzoquinone (CMQ) . A most preferred compound is ARQ. Also provided is a pharmaceutical compositions comprising (a) any of the above compounds; and (b) a pharmaceutically acceptable carrier or excipient.
Also included herein is a method of inhibiting function of mitochondrial VDAC protein and thereby inhibiting cation flux in a cell, comprising providing to the cell an effective amount of
(i) geldanamycin (GA) or a derivative thereof, or (ii) a redox-cycling benzoquinone/semiquinone/or hydroquinone moiety of GA or derivative,
(iii) a salt of any of (i) or (ii),
(iv) a pharmaceutically acceptable salt of the GA or derivative(i), the redox- cycling moiety of (iii), or (v) a pharmaceutical composition that comprises any of (i) - (iv), which GA, derivative, redox-cycling moiety or salt binds to and inhibits VDAC function and cation flux at subnanomolar concentrations, and which GA or derivative includes in its structure a redox-cycling benzoquinone moiety.
Also included is a method of inhibiting a HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell, comprising providing to the cells an effective amount of the compound or pharmaceutical composition as above, wherein the compound has an IC50 of less than about 10"7 M, less than about 10"8 M, or preferably less than about 10"9 M, for inhibition of the biological activity. Such biological activity may be the induction of uPA activity in the cells, or growth, scatter or invasion of the cells, wherein such invasion is of the type that results in tumor metastasis.
In another embodiment, a method for blocking calcium channels or cation flux in a cell comprises providing to the cells an effective amount of a compound or pharmaceutical composition thereof, which inhibits the activity of the mitochondrial VDAC protein which comprises providing to the cell an effective amount of a compound that is (i) geldanamycin (GA) or (ii) a derivative thereof, or (iii) a redox-cycling benzoquinone, semiquinone or /hydroquinone moiety of the GA or derivative, or (iv) a pharmaceutically acceptable salt of the GA, derivative or moiety, or a pharmaceutical composition that comprises any of (i) - (iv), which compound binds to and inhibits VDAC function and cation flux at subnanomolar concentrations.
Also provided is a method of for blocking calcium channels or cation flux in a subject, and thereby preventing or treating a disease or a condition that is preventable or treatable by such calcium channel or cation flux blockade, comprising administering to a subject in need thereof a VDAC-inhibitory effective amount of a compound that is (i) geldanamycin (GA) or (ii) a derivative thereof, or (iii) a redox-cycling benzoquinone/semiquinone/hydroquinone moiety of the GA or derivative, or (iv) a pharmaceutically acceptable salt of the GA, derivative or moiety, or a pharmaceutical composition that comprises any of (i) - (iv), which compound binds to and inhibits VDAC function and cation flux at subnanomolar concentrations. In the foregoing method, the compound is preferably one disclosed above.
In the above method the cell is preferably a human cell or the subject is a human. However, the invention is also useful in treating a non-human animal in the context of veterinary medicine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A-1D show that VDAC binds to Geldanamycin-conjugated affinity beads.
Fig. IA. GA-conjugated affinity beads were used in pull-down experiments with cell lysates from MDCK (left panel) and DBTRG cultures (right panel). Cells were treated with either 1 μM GA, 17-AAG, or under basal conditions for 24 h prior to harvest and then lysed. An aliquot of each cell lysate was incubated with GA-conjugated affinity beads. Eluates from the beads were analyzed by SDS-PAGE followed by Coomassie blue staining of the gel. Stained protein bands were excised for analysis by mass spectrometry (Table 1). Lane 1 : Molecular weight markers. Lanes 2 and 3: Total cell lysates precipitated by control and GA-conjugated affinity beads, respectively. Lanes 4 and 5: GA-conjugated affinity bead pull-downs of cell lysates from cells treated for 24 h with 1 μM 17-AAG and GA, respectively. The assignments of individual HSP90 and VDAC bands were established by mass spectrometry. Other bands identified by mass spectrometry are actins or tubulins, which have been reported as HSP90 substrates (JN Oa\ύseid et al., MolBiol Cell 5, 1265 (1994)). Fig. IB. GA-conjugated affinity bead pull-downs of HSP90 and VDAC from washed isolated mitochondria. MDCK and DBTRG washed isolated mitochondria prepared as described in the Methods section were purified by differential centrifugation according to the manufacturer's instructions (Sigma; and Materials and Methods). Lanes 1-3 are Western blot analyses of the GA affinity bead pull-down pellet fractions of the post-nuclear cell lysate (Sl) (lane 1), the mitochondrial wash supernatant (S2) (Lane T), and solubilized mitochondrial fractions (P3) (Lane 3). Lanes 4-6 are aliquots of supernatants after GA affinity bead pull-downs of Sl, S2, and solubilized P3. All preparations were subjected to immunob lotting analysis with antibodies to either HSP90 (upper half) or VDAC (lower half). Both HSP90α and VDAC were pulled down by GA affinity beads from the post-nuclear total MDCK and DBTRG cell lysate (lane 1). Only VDAC was pulled down from the first mitochondrial supernatant wash and less HSP90 was not detected after washing (lane X). HSP90 (and less VDAC) remains in the supernatant fractions after the GA-bead pull-down (lanes 4-6). VDAC enrichment occurs in the mitochondrial fraction. Fig. 1C. Free GA release of HSP90 from GA-coupled beads. GA-affmity beads were incubated with MDCK whole cell lysates at 40C for 4 h. After the beads were washed 3 times with TNSEV buffer, GA was added at the indicated concentrations for 1 h at 40C to release HSP90 and VDAC. The HSP90 and VDAC remaining on the beads were detected following the 1-h incubation by immunoblotting as described above. GA can release HSP90 from the beads after a 1-h incubation, while VDAC doesn't show any release. Fig. ID. Free GA release of VDAC from GA-coupled beads. Mitochondria purification from MDCK cells and DBTRG cells was performed by differential centrifugation (see Materials and Methods). After dissolving in TNSEV buffer, 80 μg of mitochondrial protein and 10 μg recombinant human VDAC (rhVDAC) (Y. Shi et αl, Biochem Biophys Res Commun 303, 475 (2003)) were incubated with GA beads for 4hr. This was followed by incubation with free GA at concentrations ranging from 5-80 μM at 4°C overnight. VDAC remaining on the beads after washing was measured by immunoblotting. VDAC is competed from the GA beads by 10-40 μM GA.
Figure 2 shows that VDAC and "permeability transition pore" inhibitors UbO, Decyl-Ub, H2DIDS, and CsA block HGF/SF mediated uPA-plasmin activation. MDCK cells were seeded in 96-well plates at 1500 cells/well. 24 hrs later, HGF/SF (60 μg/ml) was added to all wells (with the exception of wells used to calculate basal plasmin activation). Immediately after HGF/SF addition, compounds at different concentrations were added into wells and after another 24 hours incubation, plates were washed and 200 ml of reaction buffer were added to each well. The plates were then incubated at 37°C in 5% CO2 for 4 h and were read at a single wavelength of 405nm. CsA and H2DIDS showed activity to 100 μM, while UbO and Decyl-Ub block uPA-plasmin activation at 10 μM and 100 μM, respectively.
Figures 3A-3D show effects of the benzoquinone 17-ARQ (Figs 3 A and 3B) and the fluorinated GA derivative, 17-FEG (Figs. 3C and 3D) on HGF-stimulated biological activities in MDCK cells. Fig. 3 A shows Western blots of cell lysates from MDCK incubated with various dilutions of the benzoquinone 17-ARQ in DMSO at concentrations ranging from 10"6 to 10"12M and incubated for 24 hours . Control cells (lane 1, labeled "C") were not treated; cells in lane 2, labeled "D" were treated with the DMSO solvent only. Cells were then lysed, and processed for Western blots. Protein concentration was determined by DC protein assay (Bio-Rad). Equal quantities of protein were loaded onto gels and separated by SDS-PAGE. Proteins were transferred to PVDF membranes as shown above for Western blotting. The membranes were blocked and blotted with specific antibodies as noted above. Membranes were processed as described in the Examples. Protein bands reactive with specific antibodies for Met, HSP90, VDAC and β actin (loading control) are shown. Fig. 3B shows that ARQ inhibits uPA-plasmin activation induced by HGF in a dose dependent manner over the range of 10"5 to 10"8 M. Controls were either untreated cells or cells treated with the DMSO solvent. The assay is described in Example I. Fig. 3C shows results of treating MDCK cells with 17-FEG serially diluted with DMSO at concentrations from 10"6M to 10"9M, respectively. Incubation and processing was as above. Control (C) cells were not treated (lane 1) and additional controls (D) were treated with DMSO only (lane 2). Western blots of proteins from lysed cells were prepared as described herein. Antibodies used are those shown given above for Met, HSP90, VDAC and β-actin (loading control). Fig. 3D shows results of a uPA activity assay conducted as described in the Examples and above for Fig. 3B. Cells were exposed to HGF/SF (100ng/ml) alone or with 17-FEG at the indicated concentrations. After a 24 hr incubation, cells were processed as described and 200 μl of reaction buffer containing the plasmin-sensitive chromophore was added, and the color reactions evaluated after 2 hrs of incubation at 405 nm. Figure 4A-4H show effects of UbO, Decyl-Ub, and GA on all external cations on whole-cell current in MDCK cells treated with HGF/SF. Fig. 4A. Membrane current as a function of 300-ms voltage ramps (I-V plots) from -100 to 100 mV. NMDG+ indicates substitution of all external cations with n-methyl-D-glucamine. Cs aspartate solution in pipette. Fig. 4B. Membrane current recorded at -80 mV ( • ) and 80 mV ( o )versus time. Numerals indicate respective times corresponding to the numbered plots of current vs. voltage (I-V's) shown in Fig. 4A. NMDG+ as in Fig. 4A. Holding potential = -30 mV. Fig. 4C. Effect of ubiquinone. Membrane current as a function of 300-ms voltage ramps (I-V plots) from -100 to 100 mV. Cs aspartate solution in pipette. Fig. 4D. Membrane current recorded at -80 mV ( • ) and 80 mV ( o )versus time. Numerals indicate respective times corresponding to I- Vs shown in top. Holding potential = -30 mV. Fig. 4E. Effect of decyl-ubiquinone. Membrane current as a function of 300-ms voltage ramps from -100 to 100 mV. Cs aspartate solution in pipette. Fig. 4F. Membrane current recorded at -80 mV ( • ) and 80 mV ( o )versus time. Numerals indicate respective times corresponding to the numbered plots of current vs. voltage (I-V's) shown in Fig. 4E.
Holding potential = -30 mV. Fig. 4G. Effect of geldanamycin. Membrane current as a function of 300-ms voltage ramps from -90 to 90 mV. K gluconate solution in pipette. Fig. 4H. Membrane current recorded at -80 mV ( • ) and 80 mV ( o )versus time. Numerals indicate respective times corresponding to the numbered plots of current vs. voltage (I-V's) shown in Fig. 4G. Holding potential = -30 mV.
Figure 5 shows the chemical/schematic structure of four types of affinity beads to which different molecules have been conjugated for use in cell lysate pull-down experiments: C6-GA-beads, C12-GA beads, PEG-GA beads and C6-AQ beads.
Figure 6 shows results of an experiment in which MDCK cell lysates were pulled down with each of the four benzoquinone bead preparations in Fig. 5. Samples were analyzed by SDS PAGE WB for HSP90 (upper panel) and VDAC (lower panel). Lane 1, conventional C6-GA beads; lane 2, Ci2-GA beads; lane 3, PEG-GA beads; and lane 4, C6- AQ beads. All beads with GA effectively pulled down VDAC (only C6-AQ, a benzoquinone lacking the ansamycin ring, did not pull-down VDAC. Figure 7/1 - 7/3 is a list of GA and derivatives and benzoquinone compounds of the present invention showing chemical structures. Figs. 7/1 and 7/2 show GA and derivatives compounds (given numeric designations). Fig. 7/3 shows benzoquinone compounds derived from some of these GA compounds (given lower case letter designations). DESCRIPTION OF THE PREFERRED EMBODIMENTS
The compounds useful in this invention have the property of inhibiting (a) the activation of Met by HGF/SF in cancer cells and (b) HGF/SF-induced tumor cell invasion. The GA and preferred derivatives or salts inhibit HSP90, but also may inhibit mitochondrial activity through VDAC as well as inhibiting non-selective cation currents in cells at picomolar concentrations. The compounds preferably have an IC50 of less than about 10~8 M for inhibition of biological activity.
Also provided is method of for blocking calcium channels or cation flux in a subject, preferably a human, and thereby preventing or treating a disease or a condition that is preventable or treatable by calcium channel or cation flux blockade, which method comprises administering to a subject in need thereof an effective amount of a compound, or pharmaceutical composition thereof, as indicated herein.
Compounds that are useful in the above methods preferably are those in which VDAC inhibition equals or exceeds that of mitochondria specific ubiquinone (UbO) or decyl-ubiquinone (Decyl-Ub).
Nonlimiting examples of useful benzoquinone compounds (or their salts) that make up part of the ring structure of GA and its derivatives were listed above.
The present invention is directed to a method for inhibiting, among other activities, HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell. Such Met-mediated biological activities include the induction of uPA activity in the cells or cell growth, scatter or invasion. Such invasive activity reflects the cells' ability to metastasize in vivo. Thus, the present methods include the inhibition of tumor metastasis of a primary or recurrent tumor in a subject Preferably, the inhibition produced by the present compounds and methods results in measurable regression of the primary, recurrent or metastatic tumor or measurable attenuation of tumor growth in the subject. The method is particularly useful for the treatment of a Met-positive tumors in a susceptible or affected subject. The compound or pharmaceutical composition is administered to a subject who is either (a) at risk for development of the tumor, or (b) in the case of an already treated subject, at risk for recurrence of the tumor. The above method is also used to induce an antitumor or anticancer response in a mammal, preferably a human, which response is: (a) a partial response characterized by (i) at least a 50% decrease in the sum of the products of maximal perpendicular diameters of all measurable lesions; (ii) no evidence of new lesions, and (iii) no progression of any preexisting lesions, or (b) a complete response characterized by the disappearance of all evidence of tumor or cancer disease for at least one month.
The invention is directed to a method of inhibiting a HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell, comprising providing to the cells an effective amount of a compound as described herein that has an IC50 of less than about 10"8 M or less than about 10"9 M or less than about 10"10 M or less than about 10"11 M. The biological activity may be measured the induction of uPA activity in the cells, or patch clamp cell membrane cationic current, scatter of the cells, invasion of said cells in vitro.
A pharmaceutical composition according to this invention comprises the compounds described herein in a formulation that, as such, is known in the art.
Pharmaceutical compositions within the scope of this invention include all compositions wherein the GA derivative, preferably a benzoquinone, a semiquinone or a hydroquinone salt of the benzoquinone is contained in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 mg to 100 mg/kg/body mass, more preferably 1 mg to 100 mg/kg body mass, more preferably 10 mg - 10 mg/kg body mass.
A preferred dose of a benzoquinone or other compound of the present invention is one that is sufficient to inhibit VDAC activity, for example, a compound that has an IC50 of less than about 10"8 M for inhibition of a biological activity mediated by VDAC activity in mitochondria and cation flux in a cell..
Preferred doses are lower than doses that are typically associated with inhibition of HSP90 (Figure 3 C & D). Thus, in particular in the case of a known benzoquinone or related compound, the effective does is preferably one that inhibits uPA (Figure 3 D) and cation flux in a cell (Figure 4A-4H) and interferes with growth, invasion, scatter, etc, of such a cell. While geldanamycin drug effect on HSP90 is measured by HSP90 induction and reduction of Met (Figure 3C). As indicated, the effectiveness of the compound or pharmaceutical composition may be measured as its biological activity in an assay as described herein, in vitro or in vivo. Alternatively, the biological effectiveness may be measured by direct testing of the target parameters of the compound in the treatment of cancer in a subject, such as in any known and accepted murine or other small or large animal model, or in a human subject being treated. The effective dose or amount may be less than the amount needed to cause significant inhibition, such as >90% or >80% or >70% or >60% or >50% inhibition or of HSP90 or of a biological effect mediated by HSP90 but not one mediated by VDAC or by mechanisms that work through mitochondrial function or through inhibition of cation flux in a cell.
In addition to the pharmacologically active molecule, pharmaceutical compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically as is well known in the art. Suitable solutions for administration by injection or orally, may contain from about 0.01 to 99 percent, active compound(s) together with the excipient. The pharmaceutical preparations of the present invention are manufactured in a manner which is known, for example, by means of conventional mixing, granulating, dissolving, or lyophilizing processes. Suitable excipients may include fillers binders, disintegrating agents, auxiliaries and stabilizers, all of which are known in the art. Suitable formulations for parenteral administration include aqueous solutions of the proteins in water-soluble form, for example, water-soluble salts. Compounds are preferably be dissolved in dimethylsulfoxide (DMSO) and administered intravenously (i.v.) as a DMSO solution mixed into an aqueous i.v. formulation (see Goetz JP et al., 2005, J. Clin. Oncol. 25:1078-1087, for a description of the administration of 17-allylamino-17- demethoxygeldanamycin. The compounds may be administered orally in a different formulation. For the compounds and methods of the present invention, a preferred solvent is DMSO further diluted into a standard aqueous i.v. solution.
In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension.
The compositions may be in the form of a lyophilized particulate material, a sterile or aseptically produced solution, a tablet, an ampule, etc. Vehicles, such as water (preferably buffered to a physiologically acceptable pH, as for example, in phosphate buffered saline) or an appropriate organic solvent, other inert solid or liquid material such as normal saline or various buffers may be present. The particular vehicle is not critical, and those skilled in the art will know which vehicle to use for any particular utility described herein.
In general terms, a pharmaceutical composition is prepared by mixing, dissolving, binding or otherwise combining the polymer or polymeric conjugate of this invention with one or more water-insoluble or water-soluble aqueous or non-aqueous vehicles. It is imperative that the vehicle, carrier or excipient, as well as the conditions for formulating the composition are such that do not adversely affect the biological or pharmaceutical activity of the active compound. Those skilled in the art will know how to formulate and administer the pharmaceutical compositions and carry out the treatment methods disclosed herein. See, for example, Gennaro, AE., Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; 2003 or latest edition); Pharmaceutical Analysis, Watson D ed. 1 st Edition 1999: Harcourt Pub Limited London, UK. Pharmaceutical Calculations, Stoklosa MJ and Ansel C eds. 10 th Edition, 1996: Williams & Wilkins PA, USA; Goodman & Gilman's The Pharmacological Basis of Therapeutics, Brunton, LL et al, eds, 11th edition, McGraw-Hill Professional, New York, 2005, all of which references are incorporated herein by reference.
Subjects, Treatments Modes and Routes of Administration The preferred animal subject of the present invention is a mammal. The invention is particularly useful in the treatment of human subjects. By the term "treating" is intended the administering to subjects of a pharmaceutical composition comprising a compound as described herein, preferably a benzoquinone. Treating includes administering the agent to subjects at risk for developing a Met-positive tumor prior to evidence of clinical disease, as well as subjects diagnosed with such tumors or cancer, who have not yet been treated or who have been treated by other means, e.g., surgery, conventional chemotherapy, and in whom tumor burden has been reduced even to the level of not being detectable. Thus, this invention is useful in preventing or inhibiting tumor primary growth, recurrent tumor growth, invasion and/or metastasis or metastatic growth. The pharmaceutical compositions of the present invention wherein the compound is combined with pharmaceutically acceptable excipient or carrier, may be administered by any means that achieve their intended purpose. Amounts and regimens for the administration of can be determined readily by those with ordinary skill in the clinical art of treating any of the particular diseases. Preferred amounts are described below.
The active compounds of the invention may be administered orally, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
In general, the present methods include administration by parenteral routes, including injection or infusion using any known and appropriate route for the subject's disease and condition. Parenteral routes include subcutaneous (s.c.) intravenous (i.v.), intramuscular, intraperitoneal, intrathecal, intracisternal transdermal, topical, rectal or inhalational. Also included is direct intratumoral injection. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Preferably the active compound of the invention is administered in a dosage unit formulation containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
In one treatment approach, the compounds and methods are applied in conjunction with surgery. Thus, an effective amount of the compound is applied directly to the site of surgical removal of a tumor (whether primary or metastatic). This can be done by injection or "topical" application in an open surgical site or by injection after closure. In one embodiment, a specified amount of the compound, preferably about 1 ng-1 mg, is added to about 700 ml of human plasma that is diluted 1 : 1 with heparinized saline solution at room temperature. Human IgG in a concentration of 500 μg/dl (in the 700 ml total volume) may also be used. The solutions are allowed to stand for about 1 hour at room temperature. The solution container may then be attached directly to an iv infusion line and administered to the subject at a preferred rate of about 20 ml/min.
In another embodiment, the pharmaceutical composition is directly infused i.v. into a subject. The appropriate amount, preferably about 10 mg - 1 gram , is added to about 250 ml of heparinized saline solution and infused iv into patients at a rate of about 20 ml/min. The composition can be given one time but generally is administered six to twelve times (or even more, as is within the skill of the art to determine empirically). The treatments can be performed daily but are generally carried out every two to three days or as infrequently as once a week, depending on the beneficial and any toxic effects observed in the subject. If by the oral route, the pharmaceutical composition, preferably in a convenient tablet or capsule form, may be administered once or more daily.
The pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration, and all three types of formulation may be used simultaneously to achieve systemic administration of the active ingredient.
For lung instillation, aerosolized solutions are used. In a sprayable aerosol preparations, the active protein or small molecule agent may be in combination with a solid or liquid inert carrier material. This may also be packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant. The aerosol preparations can contain solvents, buffers, surfactants, and antioxidants in addition to the protein of the invention.
The appearance of tumors in sheaths ("theca") encasing an organ often results in production and accumulation of large volumes of fluid in the organ's sheath. Examples include (1) pleural effusion due to fluid in the pleural sheath surrounding the lung, (2) ascites originating from fluid accumulating in the peritoneal membrane and (3) cerebral edema due to metastatic carcinomatosis of the meninges. Such effusions and fluid accumulations generally develop at an advanced stage of the disease. The present invention contemplates administration of the pharmaceutical composition directly administration into cavities or spaces, e.g. , peritoneum, thecal space, pericardial and pleural space containing tumor. That is the agent is directly administered into a fluid space containing tumor cells or adjacent to membranes such as pleural, peritoneal, pericardial and thecal spaces containing tumor. These sites display malignant ascites, pleural and pericardial effusions or meningeal carcinomatosis . The drug is preferably administered after partial or complete drainage of the fluid (e.g., ascites, pleural or pericardial effusion ) but it may also be administered directly into the undrained space containing the effusion, ascites and/or carcinomatosus. In general, the compound's dose may vary from 1 nanogram to 1 mg, preferably, 10 ng to 100 μg, and given every 3 to 10 days. It is continued until there is no reaccumulation of the ascites or effusion. Therapeutic responses are considered to be no further accumulation of four weeks after the last intrapleural administration.
For topical application, the active compound may be incorporated into topically applied vehicles such as salves or ointments, as a means for administering the active ingredient directly to the affected area. Scarification methods, known from studies of vaccination, can also be used. The carrier for the active agent may be either in sprayable or nonsprayable form. Non-sprayable forms can be semi-solid or solid forms comprising a carrier indigenous to topical application and having a dynamic viscosity preferably greater than that of water. Suitable formulations include, but are not limited to, solution, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like. If desired, these may be sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like. Examples of preferred vehicles for non-sprayable topical preparations include ointment bases, e.g., polyethylene glycol- 1000 (PEG-1000); conventional creams such as HEB cream; gels; as well as petroleum jelly and the like.
Other pharmaceutically acceptable carriers according to the present invention are liposomes or other timed-release or gradual release carrier or drug delivery device known in the art
Combinations with Chemotherapeutic and Biological Anti-cancer Agents
Conventional chemotherapeutic agents can be used together with the present compounds, by any conventional route and at doses readily determined by those of skill in the art. Anti-cancer chemotherapeutic drugs useful in this invention include but are not limited to antimetabolites, anthracycline, vinca alkaloid, anti-tubulin drugs, antibiotics and alkylating agents. Representative specific drugs that can be used alone or in combination include cisplatin (CDDP), adriamycin, dactinomycin, mitomycin, carminomycin, daunomycin, doxorubicin, tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), verapamil, podophyllotoxin, 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, camptothecin, actinomycin-D, mitomycin C, aminopterin, combretastatin(s) and derivatives and prodrugs thereof.
Any one or more of such drugs, newer drugs targeting oncogene signal transduction pathways, or that induce apoptosis or inhibit angiogenesis, and biological products such as nucleic acid molecules, vectors, antisense constructs, siRNA constructs, and ribozymes, as appropriate, may be used in conjunction with the present compounds and methods. Examples of such agents and therapies include, radiotherapeutic agents, antitumor antibodies with attached anti-tumor drugs such as plant-, fungus-, or bacteria- derived toxin or coagulant, ricin A chain, deglycosylated ricin A chain, ribosome inactivating proteins, sarcins, gelonin, aspergillin, restricticin, a ribonuclease, a epipodophyllotoxin, diphtheria toxin, or Pseudomonas exotoxin. Additional cytotoxic, cytostatic or anti-cellular agents capable of killing or suppressing the growth or division of tumor cells include anti-angiogenic agents, apoptosis-inducing agents, coagulants, prodrugs or tumor targeted forms, tyrosine kinase inhibitors, antisense strategies, RNA aptamers, siRNA and ribozymes against VEGF or VEGF receptors. Any of a number of tyrosine kinase inhibitors are useful when administered together with, or after, the present compounds. These include, for example, the 4-aminopyrrolo[2,3-d]pyrimidines (U.S. Pat. No. 5,639,757). Further examples of small organic molecules capable of modulating tyrosine kinase signal transduction via the VEGF -R2 receptor are the quinazoline compounds and compositions (U.S. Pat. No. 5,792,771). Other agents which may be employed in combination with the present invention are steroids such as the angiostatic 4,9(1 l)-steroids and C21-oxygenated steroids (U.S. Pat. No. 5,972,922).
Thalidomide and related compounds, precursors, analogs, metabolites and hydrolysis products (U.S. Pat. Nos. 5,712,291 and 5,593,990) may also be used in combination to inhibit angiogenesis. These thalidomide and related compounds can be administered orally. Other anti-angiogenic agents that cause tumor regression include the bacterial polysaccharide CMlOl (currently in clinical trials) and the antibody LM609. CMlOl induces neovascular inflammation in tumors and downregulates expression VEGF and its receptors. Thrombospondin (TSP-I) and platelet factor 4 (PF4) are angiogenesis inhibitors that associate with heparin and are found in platelet α granules. Interferons and matrix metalloproteinase inhibitors (MMPFs) are two other classes of naturally occurring angiogenic inhibitors that can be used. Tissue inhibitors of metalloproteinases (TIMPs) are a family of naturally occurring MMPFs that also inhibit angiogenesis. Other well- studied anti-angiogenic agents are angiostatin, endostatin, vasculostatin, canstatin and maspin.
Chemotherapeutic agents are administered as single agents or multidrug combinations, in full or reduced dosage per treatment cycle. The combined use of the present compositions with low dose, single agent chemotherapeutic drugs is particularly preferred. The choice of chemotherapeutic drug in such combinations is determined by the nature of the underlying malignancy. For lung tumors, cisplatin is preferred. For breast cancer, a microtubule inhibitor such as taxotere is the preferred. For malignant ascites due to gastrointestinal tumors, 5-FU is preferred. "Low dose" as used with a chemotherapeutic drug refers to the dose of single agents that is 10-95% below that of the approved dosage for that agent (by the U.S. Food and Drug Administration, FDA). If the regimen consists of combination chemotherapy, then each drug dose is reduced by the same percentage. A reduction of >50% of the FDA approved dosage is preferred although therapeutic effects are seen with dosages above or below this level, with minimal side effects. Multiple tumors at different sites may be treated by systemic or by intrathecal or intratumoral administration of the benzoquinone and other compounds described herein.
The optimal chemotherapeutic agents and combined regimens for all the major human tumors are set forth in Bethesda Handbook of Clinical Oncology, Abraham J et al., , Lippincott William & Wilkins, Philadelphia, PA (2001); Manual of Clinical Oncology, Fourth Edition, Casciato, DA et al., Lippincott William & Wilkins, Philadelphia, PA (2000) both of which are herein incorporated in entirety by reference. In Vivo Testing of Candidate Compounds
The present compounds may be tested for therapeutic efficacy in well established rodent models which are considered to be representative of a human tumor. The overall approach is described in detail in Geran, R.I. et ah, "Protocols for Screening Chemical Agents and Natural Products Against Animal Tumors and Other Biological Systems (3d Ed)", Cane. Chemother. Reports, Part 3, 5:1-112; and Plowman, J et ah, In: Teicher, B, ed., Anticancer Drug Development Guide: Preclinical Screening, Clinical Trials and Approval, Part II: In Vivo Methods, Chapter 6, "Human Tumor Xenograft Models in NCI Drug Development," Humana Press Inc., Totowa, NJ, 1997. See also, Talmadge, JE et al, "Murine Models to Evaluate Novel and Conventional Therapeutic Strategies for Cancer" Amer J Pathol. 2007, 170:793. These references are hereby incorporated by reference in their entirety. Human Tumor Xenograft Models The preclinical discovery and development of anticancer drugs as implemented by the National Cancer Institute (NCI) consists of a series of test procedures, data review, and decision steps (Grever, MR, Semin Oncol., 7P:622-638 (1992)). Test procedures are designed to provide comparative quantitative data, which in turn, permit selection of the best candidate agents from a given chemical or biological class. Below, we describe human tumor xenograft systems, emphasizing melanomas, that are currently employed in preclinical drug development.
Since 1975, the NCI approach to drug discovery involved prescreening of compounds in the i.p. -implanted murine P388 leukemia model (see above), followed by evaluation of selected compounds in a panel of transplantable tumors (Venditti, J.M. et al., In: Garrattini S et al., eds., Adv. Pharmacol and Chemother 2:1-20 (1984)) including human solid tumors. The latter was made possible through the development of immunodeficient athymic nude (nu/nu) mice and the transplantation into these mice of human tumor xenografts (Rygaard, J. et al., Acta Pathol. Microbiol. Scand. 77:758-760 (1969); Giovanella, G.C. et al, J. Natl Cane. Inst. 57:615-619 (1973)). Studies assessing the metastatic potential of selected murine and human tumor-cell lines (B16, A-375, LOX- IMVI melanomas, and PC-3 prostate adenocarcinoma) and their suitability for experimental drug evaluation supported the importance of in vivo models derived from the implantation of tumor material in anatomically appropriate host tissues; such models are well suited for detailed evaluation of compounds that inhibit activity against specific tumor types. Beginning about 1990, the NCI began employing human tumor cell lines for large-scale drug screening ((Boyd, MR, In: DeVita, VT et al, Cancer: Principles and Practice of Oncology, Updates, vol 3, Philadelphia, Lippinicott, 1989, pp 1-12; Plowman, supra). Cell lines derived from seven cancer types (brain, colon, leukemia, lung, melanoma, ovarian, and renal) were acquired from a wide range of sources, frozen, and subjected to a battery of in vitro and in vivo characterization. This approach shifted the screening strategy from "compound-oriented" to "disease-oriented" drug discovery (Boyd, supra). Compounds of identified by the screen, demonstrating disease-specific, differential cytotoxicity were considered "leads" for further preclinical evaluation. A battery of human tumor xenograft models was created to deal with such needs. The initial solid tumors established in mice are maintained by serial passage of 30-
40 mg tumor fragments implanted s.c. near the axilla. Xenografts are generally not utilized for drug evaluation until the volume-doubling time has stabilized, usually around the fourth or fifth passage.
The in vivo growth characteristics of the xenografts determine their suitability for use in the evaluation of test agent antitumor activity, particularly when the xenografts are utilized as early stage s.c. models. As used herein, an early stage s.c. model is defined as one in which tumors are staged to 63-200 mg prior to the initiation of treatment. Growth characteristics considered in rating tumors include take -rate, time to reach 200 mg, doubling time, and susceptibility to spontaneous regression. As can be noted, the faster- growing tumors tend to receive the higher ratings.
Any of a number of transgenic mouse models known in the art can be used to test the present compounds. A particularly useful murine human HGF/SF transgenic model has been described by one of the present inventors and his colleagues and may be used to test the present compounds against human tumor xenografts in vivo. See, Zhang YW et al. (2005) Oncogene 24:101-106; U.S. Pat.App Ser. No. 60/587,044, which references are incorporated by reference in their entirety. Other longer- known models are described below.
Advanced-Stage Subcutaneous Xenograft Models
Such s. c. -implanted tumor xenograft models are used to evaluate the antitumor activity of test agents under conditions that permit determination of clinically relevant parameters of activity, such as partial and complete regression and duration of remission (Martin DS et al, Cancer Treat Rep (55:37-38 (1984); Martin DS et ah, Cancer Res. 46:2189-2192 (1986); Stolfϊ, RL et al., J. Natl Cane Inst 80:52-55 (1988)). Tumor growth is monitored and test agent treatment is initiated when tumors reach a weight range of 100- 400 mg (staging day, median weights approx. 200 mg), although depending on the xenograft, tumors may be staged at larger sizes. Tumor sizes and body weights are obtained approximately 2 times/wk. Through software programs (developed by staff of the Information Technology Branch of DTP of the NCI), data are stored, various parameters of effects are calculated, and data are presented in both graphic and tabular formats. Parameters of toxicity and antitumor activity are defined as follows:
1. Toxicity: Both drug-related deaths (DRD) and maximum percent relative mean net body weight losses are determined. A treated animal's death is presumed to be treatment-related if the animal dies within 15 d of the last treatment, and either its tumor weight is less than the lethal burden in control mice, or its net body weight loss at death is 20% greater than the mean net weight change of the controls at death or sacrifice. A DRD also may be designated by the investigator. The mean net body weight of each group of mice on each observation day is compared to the mean net body weight on staging day. Any weight loss that occurs is calculated as a percent of the staging day weight. These calculations also are made for the control mice, since tumor growth of some xenografts has an adverse effect on body weight.
2. Optimal % T/C: Changes in tumor weight (A weights) for each treated (T) and control (C) group are calculated for each day tumors are measured by subtracting the median tumor weight on the day of first treatment (staging day) from the median tumor weight on the specified observation day. These values are used to calculate a percent T/C as follows:
% T/C = (ΔT/ΔC) χ 100 where ΔT>0 or
= (ΔT/Tι) x 100 where ΔT<0 (1 ) and Ti is the median tumor weight at the start of treatment. The optimum (minimum) value obtained after the end of the first course of treatment is used to quantitate antitumor activity. 3. Tumor growth delay: This is expressed as a percentage by which the treated group weight is delayed in attaining a specified number of doublings; (from its staging day weight) compared to controls using the formula:
[(T - C)/C] x 100 (2) where T and C are the median times (in days) for treated and control groups, respectively, to attain the specified size (excluding tumor- free mice and DRDs). The growth delay is expressed as percentage of control to take into account the growth rate of the tumor since a growth delay based on (T - C) alone varies in significance with differences in tumor growth rates. 4. Net log cell kill: An estimate of the number of logio units of cells killed at the end of treatment is calculated as:
{[(T - C) - duration of treatment] x 0.301 / median doubling time} (3) where the "doubling time" is the time required for tumors to increase in size from 200 to 400 mg, 0.301 is the logio of 2, and T and C are the median times (in days) for treated and control tumors to achieve the specified number of doublings. If the duration of treatment is 0, then it can be seen from the formulae for net log cell kill and percent growth delay that log cell kill is proportional to percent growth delay. A log cell kill of 0 indicates that the cell population at the end of treatment is the same as it was at the start of treatment. A log cell kill of +6 indicates a 99.9999% reduction in the cell population.
5. Tumor regression: The importance of tumor regression in animal models as an end point of clinical relevance has been propounded by several investigators (Martin et al., 1984, 1986 supra; Stolfi et al., supra). Regressions are defined-as partial if the tumor weight decreases to 50% or less of the, tumor weight at the start of treatment without dropping below 63 mg (5 x 5 mm tumor). Both complete regressions (CRs) and tumor free survivors are defined by instances in which the tumor burden falls below measurable limits (<63 mg) during the experimental period. The two parameters differ by the observation of either tumor regrowth (in CR animals) or no regrowth (=tumor- free) prior to the final observation day. Although one can measure smaller tumors, the accuracy of measuring a s.c. tumor smaller than 4 x 4 mm or 5 x 5 mm (32 and 63 mg, respectively) is questionable. Also, once a relatively large tumor has regressed to 63 mg, the composition of the remaining mass may be only fibrous material/scar tissue. Measurement of tumor regrowth following cessation of treatment provides a more reliable indication of whether or not tumor cells survived treatment. Most xenografts that grow s.c. may be used in an advanced-stage model, although for some tumors, the duration of the study may be limited by tumor necrosis. As mentioned previously, this model enables the measurement of clinically relevant parameters and provides a wealth of data on the effects of the test agent on tumor growth. Also, by staging day, the investigator is ensured that angiogenesis has occurred in the area of the tumor, and staging enables "no-takes" to be eliminated from the experiment. However, the model can be costly in terms of time and mice. For slower-growing tumors, the passage time required before sufficient mice can be implanted with tumors may be at least ~4 wks, and an additional 2-3 wks may be required before the tumors can be staged. To stage tumors, more mice (as many as 50-100% more) than are needed for actual drug testing must be implanted. Early Treatment and Early Stage Subcutaneous Xenograft Models
These models are similar to the advanced-stage model, but, because treatment is initiated earlier in the development of the tumor, useful tumors are those with > 90% take- rate (or < 10% spontaneous regression rate). The "early treatment model" is defined as one in which treatment is initiated before tumors are measurable, i.e., <63 mg. The "early stage" model as one in which treatment is initiated when tumor size ranges from 63-200 mg. The 63-mg size is used because it indicates that the original implant, about 30 mg, has demonstrated some growth. Parameters of toxicity are the same as those for the advanced-stage model; parameters of antitumor activity are similar. %T/C values are calculated directly from the median tumor weights on each observation day instead of being measured as changes (Δ) in tumor weights, and growth delays are based on the days after implant required for the tumors to reach a specified size, e.g., 500 or 1000 mg. Tumor-free mice are recorded, but may be designated as "no-takes" or spontaneous regressions if the vehicle-treated control group contains >10% mice with similar growth characteristics. A "no-take" is a tumor that fails to become established and grow progressively. A spontaneous regression (graft failure) is a tumor that, after a period of growth, decreases to ≤ 50% of its maximum size. Tumor regressions are not normally recorded, since they are not always a good indicator of antineoplastic effects in the early stage model. A major advantage of the early treatment model is the ability to use all implanted mice, which is why a good tumor take-rate is required. In practice, the tumors most suitable for this model tend to be the faster-growing ones. Challenge Survival Models
In another approach, the effect of human tumor growth on the lifespan of the host is determined. All mice dying or sacrificed owing to a moribund state or extensive ascites prior to the final observation day are used to calculate median day of death for treated (T) and control (C) groups. These values are then used to calculate a percent increase in life span ("ILS") as follows: % ILS = [(T - C/C] x 100 (4)
Where possible, titration groups are included to establish a tumor doubling time for use in logio cell kill calculations. A death (or sacrifice) may be designated as drug-related based on visual observations and/or the results of necropsy. Otherwise, treated animal deaths are-designated as treatment-related if the day of death precedes the mean day of death of the controls (-2SD) or if the animal dies without evidence of tumor within 15 days of the last treatment.
Response of Xenograft Models to Standard Agents
In obtaining drug sensitivity profiles for the advanced-stage s.c. xenograft models, the test agent is evaluated following i.p. administration at multiple dose levels. The activity ratings are based on the optimal effects attained with the maximally tolerated dose (<LD2o) of each drug for a given treatment schedule which is selected on the basis of the doubling time of a given tumor, with longer intervals between treatments for slower growing tumors. As described in Plowman, J. et ah, supra, at least minimal antitumor effects (%T/C
≤ 40) were produced in the melanoma group by at least 2, and as many as 10, clinical drugs. The number of responses appeared to be independent of doubling time and histological type with a range in the number of responses observed for tumors (seen in each subpanel of other tumor types as well). When the responses are considered in terms of the more clinically relevant end points of partial or complete tumor regression, these tumors models (across all tumors) were quite refractory to standard drug therapy; the tumors did not respond to any of the drugs tested in 30 of 48 (62.5%) of all tumors. Strategy for Initial Compound Evaluation In Vivo
The in vitro primary screens provide a basis for selecting the most appropriate tumor lines to use for follow-up in vivo testing, with each compound tested only against xenografts derived from cell lines demonstrating the greatest sensitivity to the agent in vitro. The early strategy for in vivo testing emphasized the treatment of animals bearing advanced-stage tumors. Based on the specific information available to guide dose selection here, much lower doeses than those used for typical test agents are selected. Single mice are preferably treated with single ip bolus doses of between 1 pg/kg and and 1 mg/kg and observed for 14 d. Sequential 3-dose studies may be conducted as necessary until a nonlethal dose range is established. The test agent is then evaluated preferably in three s.c. xenograft models using tumors that are among the most sensitive to the test agent in vitro and that are suitable for use as early stage models. The compounds are administered ip, as suspensions if necessary, on schedules based, with some exceptions, on the mass doubling time of the tumor. For example, for doubling times of 1.3-2.5, 2.6-5.9, and 6-10 d, preferred schedules are: daily for five treatments (qd x 5), every fourth day for three treatments (q4d x 3), and every seventh day for three treatments (q7d x 3). For most tumors, the interval between individual treatments approximates the doubling time of the tumors, and the treatment period allows a 0.5-1.0 logio unit of control tumor growth. For tumors staged at 100-200 mg, the tumor sizes of the controls at the end of treatment should range from 500-2000 mg, which allows sufficient time after treatment to evaluate the effects of the test agent before it becomes necessary to sacrifice mice owing to tumor size. Detailed Drug Studies Once a compound has been identified as demonstrating in vivo efficacy in initial evaluations, more detailed studies are designed and conducted in human tumor xenograft models to explore further the compound's therapeutic potential. By varying the concentration and exposure time of the tumor cells and the host to the drug, it is possible to devise and recommend treatment strategies designed to optimize antitumor activity. The importance of "concentration x time" on the antitumor effects of test agents were well illustrated by data obtained with amino-20M-camptothecin (Plowman, J. et ah, 1997 ', supra). Those results indicated that maintaining the plasma concentration above a threshold level for a prolonged period of time was required for optimal therapeutic effects.
Xenograft Model of Metastasis The compounds of this invention are also tested for inhibition of late metastasis using an experimental metastasis model such as that described by Crowley, CW. et al, Proc. Natl. Acad. ScL USA 90 5021-5025 (1993)). Late metastasis involves the steps of attachment and extravasation of tumor cells, local invasion, seeding, proliferation and angiogenesis. Human melanoma cells trans fected with a reporter gene, preferably the green fluorescent protein (GFP) gene, but as an alternative with a gene encoding the enzymes chloramphenicol acetyl-transferase (CAT), luciferase or LacZ, are inoculated into nude mice. This permits utilization of either of these markers (fluorescence detection of GFP or histochemical colorimetric detection of enzymatic activity) to follow the fate of these cells. Cells are injected, preferably iv, and metastases identified after about 14 days, particularly in the lungs but also in regional lymph nodes, femurs and brain. This mimics the organ tropism of naturally occurring metastases in human melanoma. For example, GFP-expressing melanoma cells (106 cells per mouse) are injected i.v. into the tail veins of nude mice. Animals are treated with a test composition at lOOμg/animal/day given q.d. IP. Single metastatic cells and foci are visualized and quantitated by fluorescence microscopy or light microscopic histochemistry or by grinding the tissue and quantitative colorimetric assay of the detectable label. Representative mice are subjected to histopatho logical and immunocytochemical studies to further document the presence of metastases throughout the major organs. Number and size (greatest diameter) of the colonies can be tabulated by digital image analysis, e.g., as described by Fu, Y. S. et al., Anat. Quant. Cytol. Histol. 77:187-195 (1989)).
For determination of colonies, explants of lung, liver, spleen, para-aortic lymph nodes, kidney, adrenal glands and s.c. tissues are washed, minced into pieces of 1-2 mm3 and the pieces pulverized in a Tekman tissue pounder for 5 min. The pulverized contents are filtered through a sieve, incubated in a dissociation medium (MEM supplemented with 10% FCS, 200 U/ml of collagenase type I and 100 μg/ml of DNase type I) for 8 hr at 370C with gentle agitation. Thereafter, the resulting cell suspension is washed and resuspended in regular medium {e.g., MEM with 10% FCS supplemented with the selecting antibiotic (G-418 or hygromycin). The explants are fed and the number of clonal outgrowths of tumor cells is determined after fixation with ethanol and staining with an apprpriate ligand such as a monoclonal antibody to a tumor cell marker. The number of colonies is counted over an 80-cm2 area. If desired, a parallel set of experiments can be conducted wherein clonal outgrowths are not fixed and stained but rather are retrieved fresh with cloning rings and pooled after only a few divisions for other measurements such as secretion of collagenases (by substrate gel electrophoresis) and Matrigel invasion. Matrigel invasion assays are described herein, though it is possible to use assays described by others (Hendrix, M.J.C. et al, Cancer Lett., 38: 137-147 (1987); Albini, A. et al, Cancer Res., 47 3239-3245 (1987); Melchiori, A., Cancer Res. 52:2353-2356 (1992)).
All experiments are performed with groups that preferably have 10 mice. Results are analyzed with standard statistical tests. Depending on the tumor, i.v. injections of 0.2-10 x 105 tumor cells 1 week after an s.c. flank injection of an equal number of tumor cells followed by an additional 5-week interval yielded a ratio of hematogenous: spontaneous pulmonary metastases and an overall pulmonary tumor burden that is convenient for evaluation. The model may peroit retrieval of numerous extrapulmonary metastatic clones from spleen, liver, kidneys, adrenal gland, para-aortic lymph nodes and s.c. sites, most of which likely represent spontaneous metastases from the locally growing tumor.
Treatment Procedure
Doses of a composition under test are determined as using, inter alia, appropriate animal models of the tumor of cancer of interest. A pharmaceutical composition of the present invention is administered. A treatment consists of injecting the subject with, for example, 1, 100 and 1000 ng of the compound intravenously in 200 ml of normal saline over a one-hour period. Treatments are given 3x/week for a total of 12 treatments. Patients with stable or regressing disease are treated beyond the 12th treatment. Treatment is given on either an outpatient or inpatient basis as needed. Patient Evaluation
Assessment of response of the tumor to the therapy is made once per week during therapy and 30 days thereafter. Depending on the response to treatment, side effects, and the health status of the patient, treatment is terminated or prolonged from the standard protocol given above. Tumor response criteria are those established by the International Union Against Cancer and are listed below.
Figure imgf000026_0001
See, for example, DeVita, V. T. et al, (eds), Cancer: Principles and Practice of
Oncology, 7th Edition, Lippincott Williams & Wilkins; 2004); and Frei III, E., "Clinical trials of antitumor agents: experimental design and timeline considerations," Cancer J Sci Am., 1997, 5:127-36, which are incorporated by reference.
The efficacy of the therapy in a patient population is evaluated using conventional statistical methods, including, for example, the Chi Square test or Fisher's exact test. Long-term changes in and short term changes in measurements can be evaluated separately.
For a GA derivative compound, particularly a benzoquinone thereof, to be useful in accordance with this invention, it should demonstrate activity at the nanomolar level in at least one of the in vitro, biochemical, or molecular assays described herein and also have potent antitumor activity in vivo.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified. EXAMPLE I Materials and Methods Cell lines and drugs. MDCK (canine kidney epithelial cells) and DBTRG (human glioblastoma multiforme cells) were obtained from the American Type Culture Collection. Both cell lines were grown in Dulbecco's modified Eagle's Medium (DMEM' Gibco™, Invitrogen Corp.) and supplemented with 10% fetal bovine serum (FBS; from Hyclone) as previously described (Xie et al., supra). Geldanamycin and its chemical derivatives, including 17-(Λ/-allylamino)-17-demethoxygeldanamycin (17-AAG), 17-(2- fluoroethyl)amino-17-demethoxygeldanamycin (17-FEG or FEG) and 17-allylamino-17- demethoxygeldanamycin (17-AAG) were provided by the National Cancer Institute (CW Gourlay et al. , Nat Rev MoI Cell Biol 6, 583 (2005)) or were synthesized as described elsewhere (Shen et al., supra). Radicicol, UbO, Decyl-Ub, cyclosporine A, and H2DIDS were purchased from
Sigma. All compounds were first diluted in dimethyl sulfoxide (DMSO) at 10"2 M, separated into small aliquots (5 μl), and kept at -80° C until use. Working stocks were prepared by serial dilution in DMSO. Final dilutions added to cells were 1 : 1000 in DMEM-FBS. Preparation of GA or Quinones Immobilized to Affinity Beads - Solid-Phase
GA-Binding Assays
C6-GA and TEG-GA coupled gel-affinity beads (Fig. 4/5), as well as C6 5- acetomido-2-methoxy-3-methylbenzoquinone (quinone) were prepared as previously described (Shen et al., supra). Briefly, quinone or GA (1.5 equivalents to one affinity gel bead equivalent) were stirred with the diamine linker 1 ,6-diaminohexane or tetraethylene glycol diamine (TEG) at 5-10 equivalents of TEG to one equivalent of quinone or GA in chloroform at room temperature. Upon the complete conversion of the starting material (monitored by thin layer chromatography), the mixture was washed sequentially with diluted aqueous NaOH and brine. The organic layer was dried over anhydrous K2HCO3 , and concentrated to give a dark purple solid. ( 1H NMR obtained for each compound was consistent with the known structure.) The intermediate was then taken up in DMSO and stirred with Affi-Gel 10 beads (Bio-Rad) for two hours. The resulting purple GA-beads were washed with DMSO. Ci2-GA beads (Fig. 4/5) were prepared as follows: 17-[12-
(Boc)aminododecylamine]-17-demethoxygeldanamycine (1.5 equivalents to one affinity gel bead equivalent) was dissolved in chloroform and treated with trifluoroacetic acid for one hour. The mixture was washed with aqueous Na2HCOs solution, dried over anhydrous K2HCO3, and concentrated to give 17-(12-aminododecylamine)-17- demethoxygeldanamycine as a dark purple solid (1H NMR obtained was consisted with structure.) The intermediate was taken up in DMSO and stirred with Affϊ-Gel 10 beads (Bio-Rad) for 2 hrs. The resulting purple beads were washed thoroughly with DMSO.
Control beads were made by conjugating the affinity gel bead material with a short -chain analogue that has no affinity for HSP90. Affi-Gel 10 beads (Bio-Rad) were stirred with JV-(6-aminohexyl) acetamide (M. Zoratti et ah, Biochim Biophys Acta 1241, 139 (1995)) (1.3 equivalents) in DMSO at room temperature for 2 h, then washed thoroughly with DMSO.
The GA-conjugated and control affinity beads were washed in five volumes of TNESV buffer (50 mM Tris-HCl, pH 7.5, 20 mM Na2MoO4, 1% Triton X-IOO, 150 mM NaCl, and 1 mM Na3VO4) three times and rotated overnight in TNESV at 40C to hydrolyze any unreacted iV-hydroxysuccinimide, then rocked in 1% BSA in TNESV (1 :10) at room temperature for at least 3 h. After washing with TNESV three additional times, beads were resuspended in 50% TNESV and stored at -8O0C. To perform affinity bead pull-down experiments with cell lysates, 106 cells were first seeded in 100 x 20mm dishes. After cells grew to 80% confluence, cells were further incubated with or without 1 μM GA or 17-AAG (final concentration). After 24 h, cells were washed twice with PBS and lysed in TNESV buffer supplemented with Complete™ proteinase inhibitors (Roche Molecular Biochemicals). Protein concentration was determined by DC protein assay (Bio-Rad). To eliminate nonspecific binding, cell lysates were first subjected to a "pre-clearing" pull-down with control beads. The pre-cleared supernatant fractions were then subjected to pull-down with either control or GA- conjugated beads. For pull-down assays, 50 μl of control or GA-conjugated beads were added, adjusted to equal protein concentrations of lysate in 500 μl of extract buffer and rotated at 40C overnight. Beads were recovered by low speed centrifugation and washed three times with TNESV. Sixty μl of 2X sample buffer was added and the beads were boiled for 10 min. Candidate proteins in SDS-PAGE gels were stained with Coomassie blue (Invitrogen); each band was analyzed by mass spectrometry for protein identification. For target validation, protein aliquots from mitochondrial lysates or GA-affmity bead pulldown experiments were loaded and separated on 12% SDS-PAGE gels and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen). Following blocking of the membrane with 5% powdered milk solution, membranes were incubated with specific antibodies. The antibody used to detect proteins were as follows:
(a) for Met - Met 25H(from Cell Signaling),
(b) for HSP90 - a rabbit polyclonal anti-HSP90 antibody (NeoMarkers),
(c) for VDAC - an antibody made in the inventors' institution of an antibody from Alexis. (d) for β-actin - the commercial AC- 15 antibody ab6276 from Abeam which served as a loading control. After washing and further incubation with secondary antibodies conjugated to horse radish peroxidase (HRP), the membranes were incubated Enhanced Chemiluminescence (ECL) reagents (Amersham Biosciences) and the resulting chemiluminescence signal intensity was captured on radiographic film. In the study shown in Figure 3A and 3B, 5xlO5 cells were seeded in 10 cm dishes and grew until 60% confluency. 17-ARQ serially diluted with DMSO was added to the appropriate dishes at different concentrations ranging from 10"6 to 10"12M as shown in Fig. 3 A, and incubated for 24 hours . Final DMSO concentration was limited to 0.1%. Control cells (lane 1, labeled "C") were not treated; cells in lane 2, labeled "D" were treated with the DMSO solvent only. Cells were then lysed, and protein concentration was determined by DC protein assay (Bio-Rad). Equal quantities of protein were loaded onto gels and separated by SDS-PAGE. Proteins were transferred to PVDF membranes as shown above for Western blotting. The membranes were blocked and blotted with specific antibodies as noted above. Membranes were processed as above. Mass Spectrometry Analysis
Excised gel bands were digested in-gel with trypsin in a ProGest® robot (Genomic Solutions) following the procedure described by Shevchenko et al. (Anal Chem 68, 850 (1996)). Extracted peptides were analyzed on a Waters/Micromass Q-Tof API mass spectrometer equipped with a Waters/Micromass CapLC HPLC. Sample was loaded onto a Michrom Cap Trap concentrating/desalting trap column installed on a 10-port switching valve (Valco-Vici) at a 30 μl/min flow rate. After sample loading, the valve was switched to allow nanoflow reverse phase gradient elution using 0.1% formic acid in water for buffer A and 0.1% formic acid in 90% acetonitrile for buffer B. Gradient elution at an approximate 250 nl/min flow rate was achieved via flow- splitting through a stainless-steel cross (Valco-Vici). Analytical separation of peptides was performed on a 75 μm ID x 15 cm PicoFrit® column (New Objectives) packed in-house (Michrom C- 18 Magic® 5μm particles). The Q-Tof was operated in DDA mode for automated MS to MS/MS switching. Data was processed using Waters/Micromass Masslynx® v3.5 software, and automated protein identification was achieved through database searching with MS/MS data using Matrix Science Mascot Daemon software® ( DN Perkins et al., Electrophoresis 20, 3551 (1999)) and the NCBI non-redundant protein database. Mascot database search parameters included Type of search = MS/MS Ion Search, Enzyme = Trypsin, Variable modifications = Carbamidomethyl (C) JVlass values = Monoisotopic^Protein Mass = Unrestricted, Peptide Mass Tolerance = ± 2 Da, Fragment Mass Tolerance = ± 0.1 Da,Max Missed Cleavages = 2, Instrument type = ESI-QUAD-TOF. Results are presented in Table 1. Recombinant Human VDAC (rhVDAC) Expression and Purification
The DNA encoding amino acids 1-283 of human VDAC (rhVDAC-1) was isolated from human ovary cDNA (Human Ovary Quick-clone cDNA, Clonetech) by PCR using as primers:
5 ' -TATCATATGGCTGTGCCACCC ACG TATGCC-3' (SEQ ID NO: 1); and 5'-CCGCrCG^GTGCTTGAAATTCCAGTCCTAGACC-S' (SEQ ID NO:2) to introduce an in-frame 5' Ndel site (underlined), Xhol site (underlined and italicized) without a preceding stop codon. The PCR product was cloned into a pGEM-T Easy® Vector (Promega) and then subcloned into a pET-21a expression vector (Novagen). The rhVDAC-l-pET-21a construct was transformed into BL21(DE3) and expression of the His-tagged rh VD AC-I was induced by 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 4hr at 37°C with vigorous shaking. Protein purification was based upon a published method (Y. Shi et al., Biochem Biophys Res Commun 303, 475 (2003)).
Briefly, the biomass was suspended in buffer A (20 mM Tris, pH 7.9, 500 mM NaCl, and 1 mM PMSF) and homogenized by pulse sonication on ice. The inclusion bodies were incubated in 10 ml binding buffer with 1 mg/ml lysozyme for 15 min and homogenized by sonication once again. After centrifugation at 7000 rpm for 15 min, pellet was solubilized with 6 M guanidine-HCl in buffer A for 3 h with gentle stirring, followed by adjusting guanidine-HCl to 4M for another 1 hour with gentle stirring at 4°C. The supernatant containing the denatured rh VD AC-I was collected by centrifugation at 18,000χg for 20 min. Two ml of Nico beads (Novagene) pre-equilibrated with buffer B (4M guanidine-HCl, 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, and 10% glycerol) were loaded with 10 ml denatured protein supernatant, followed by washes with 5 volumes of buffer B. Further three-step washes with 3 volumes of buffer B mixed with buffer C (2% OG, 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, and 10% glycerol) plus 10 mM imidazole (B:C=1 :3, 1 :7, and 1 :15, respectively) was performed to ensure the maximal refolding and renaturing of the protein and to remove excessive impurity. Additional washes with 10 volumes of buffer C plus 10 mM imidazole were done to remove excessive guanidine before elution of His-tagged rh VD AC-I with 4 bed volumes of buffer C supplemented with 100 mM imidazole. All the above procedures were performed at 4°C. Protein purity was verified by 12% SDS-PAGE gel as well as by immunob lotting analysis.
Mitochondrial Isolation and GA-Coupled Bead Precipitation Mitochondria were isolated from cell lysates by using a commercial isolation kit
(PIERCE Chemicals) according to the manufacturer's instructions and by differential centrifugation. After centrifugation of lysate at 700 x g for 10 min at 4°C to remove nuclei and cell debris (Pl), the post-nuclear supernatant fraction (Sl) was collected and centrifuged at 12,000 x g for 15 min to harvest crude mitochondria (P2). The P2 pellet was resuspended and centrifuged again at 12,000 x g for 5 min to collect washed mitochondria (P3) and the postmitochondrial supernatant fraction (S2). The washed mitochondrial pellet was lysed with TNESV supplemented with 1% Triton X-100 and Complete™ proteinase inhibitors (Roche Molecular Biochemicals). GA-conjugated affinity bead pull-down assays followed by immunoblotting analysis with antibodies directed against HSP90 and VDAC were performed as described above.
Release of HSP90 and VDAC from GA-Conjugated Beads with Free GA
To displace bound proteins from the GA-conjugated affinity beads, MDCK total cell lysate (equivalent to 500 μg total protein for each sample) was first adjusted to 500 μl with TNSEV buffer. Twenty μl of GA-conjugated beads were added to each sample, and the suspensions were rotated at 40C for 4hr. Beads were collected by centrifugation at
4500 rpm for 15 min, washed three times with binding buffer, and re-suspended in 500 μl TMSEV buffer containing free GA at varying concentrations. After incubation for lhr at
4°C, beads were collected and washed 3x with TNSEV buffer. Fifty-five μl of SDS- loading buffer was added to washed beads, suspensions were boiled for 15 min, and 35 μl of each eluate was subjected to SDS-PAGE followed by immunoblotting analysis to detect both HSP90 and VDAC. To release mitochondrial proteins or purified rhVDAC bound to GA-conjugated beads, samples of washed mitochondria equivalent to 80 μg total protein (isolated from MDCK or DBTRG cells) or 10 μg purified rhVDAC protein were each adjusted to 500 μl TNSEV buffer followed by GA-conjugated affinity bead pull-downs for 4hr at 4°C as described above. To release VDAC, free GA was added into each sample at concentrations ranging from 0 to 80 μM and suspensions were rotated at 4°C overnight. The next day, beads were washed three times with TNSEV buffer and then boiled in SDS- loading buffer; eluted VDAC was detected by immunoblotting analysis as described above.
The HGF/SF-Met-uPA-Plasmin Cellular Assay
Cells were seeded in 24-well plates at 5000 cells/well and grown overnight in DMEM/10% FBS as previously described (Webb et ah, supra). Drugs were serially diluted from stock concentrations from l;1000 in DMEM/10% FBS media and added to the wells. A 1 : 100 dilution of the HGF -neutralizing antibody was added to the relevant wells as a standard control on each microplate. Immediately after drug or reagent addition, HGF/SF (60 units/ml) was added to all wells (with the exception of wells used to calculate basal growth and plasmin activation). Twenty- four hrs after drug/HGF/SF addition, plates were processed for the determination of plasmin activity as follows. Wells were washed twice with DMEM (without phenol red; Life Technologies, Inc.) and 200 ml of reaction buffer (50% [v/v] 0.05 units/ml plasminogen in DMEM [without phenol red], 40% [v/v] 50 mM Tris buffer [pH 8.2], and 10% [v/v] 3 mM Chromozyme PL in 100 mM glycine solution) were added to each well. The plates were then incubated at 37°C in 5% CO2 for 2hr, at which time the color generated was read as absorbance using an automated spectrophotometric plate reader at a single wavelength of 405nm.
Whole Cell Voltage-Clamp Electrophysiology
MDCK cells were cultured on 5 x 5-mm plastic coverslips and were transferred to a microperfusion chamber on the stage of an inverted microscope. Whole-cell voltage clamp was accomplished with an Axopatch 200B (Axon Instr. Co., Union City, CA) using suction-ruptured patches primarily from single cells or from cells on the edge of islands comprising 4 or 5 cells. Borosilicate glass capillaries (1.2 mm OD, 0.68 ID, type EN-I, Garner Glass Co., Claremont, CA) were used to fabricate patch pipettes (3-8 MΩ in the bath solution) with a Brown-Flaming horizontal micropipette puller (P-87, Sutter Instruments, San Rafael, CA). Patch-pipette tips were heat-polished prior to use. A micromanipulator (Narishige) fixed to the microscope was used to position the pipettes. Electrode capacitance and series resistance were compensated prior to measurements. All experiments were performed at room temperature (20 - 22°C). The standard extracellular salt solution for ion currents contained (mM): 150 NaCl, 6 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and pH adjusted to 7.4 with IN NaOH. The internal (pipette) solution for measuring ion currents contained (mM): 140 K gluconate or Cs aspartate, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES, pH 7.2 with KOH. In some experiments the external solution was exchanged for one in which cations were substituted with an impermeable organic cation (in mM): 156 JV-methyl-D-glucamine (NMDG), 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4 with 1 N HCl. Electrophysiological recordings were procured and analyzed using PClamp8® software (Axon Instr. Co, Union City, CA) and the WinASCD software (Guy Droogmans, Katholieke Universiteit, Leuven, Belgium). All results were recorded on a computer hard drive. Membrane ion conductance was computed from slopes of linear segments of the I- V plots that intersected in the voltage axis (reversal potentials).
EXAMPLE II Synthesis of Geldanamycin Benzoquinone Fragments General Methods. 1H and 13C NMR spectra were obtained on Varian Inova-600,
UnityPlus-500, VRX-500 or VRX-300 spectrometers. Mass spectra were performed by the MSU Mass Spectrometry Facility. Infrared spectra were obtained on a Matton Galaxy Series FTIR 3000 spectrophotometer. Ultraviolet-visible spectra were obtained on a Hitachi U-4001 spectrophotometer. Anhydrous solvents were purified per standard methods.
2-Methyl-4-nitroresorcinol. [Raphael, RA et al. J. Chem. Soc. Perkin Trans. 1, 1988, 1823] 1H NMR (CDCl3, 500 MHz) δ 11.30 (s, IH), 7.91 (d, J= 9.5 Hz, IH), 6.42 (d, J = 9.5 Hz, IH), 5.52 (bs, IH), 2.17 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ 161.3, 155.9, 128.1, 124.3, 112.5, 108.4, 7.9. 3-Methoxy-2-methyl-6-nitrophenol. [Raphael et ah, supra; Rinehart, KL et ah, Bioorg. Chem., 1977, 6, 353] . 1H NMR (CDCl3, 500 MHz) δ 11.10 (s, IH), 8.00 (d, J= 9.5 Hz, IH), 6.51 (d, J= 9.5 Hz, IH), 3.92 (s, 3H), 2.13 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ 164.3, 154.7, 128.2, 124.3, 114.9, 103.1, 56.1, 8.0. ό-Acetamido-S-methoxy-l-methylphenol. [Rinehart, KL et al., Bioorg. Chem., 1977, 6, 353] 1H NMR (CDCl3, 500 MHz) δ 7.95 (bs, IH), 6.74 (d, J= 9.0 Hz, IH), 6.34 (d, J = 9.0 Hz, IH), 3.76 (s, 3H), 2.13 (s, 3H), 2.12 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ 170.7, 156.8, 148.1, 119.5, 119.4, 116.3, 102.5, 55.7, 23.2, 8.8.
5-Acetamido-2-methoxy-3-methylbenzoquinone (abbreviated "AQ", MW 209.20). [Rinehart, KL et ah, Bioorg. Chem., 1977, 6, 353] 1H NMR (CDCl3, 300 MHz) δ 8.20 (bs, IH), 7.27 (s, IH), 4.06 (s, 3H), 2.18 (s, 3H), 1.88 (s, 3H). 13C NMR (CDCl3, 75 MHz) δ 184.0, 183.5, 169.2, 156.3, 137.8, 124.0, 112.5, 61.3, 24.8, 8.4.
5-Acetamido-2-(2-fluoroethyl)amino-3-methylbenzoquinone ("FEQ" - MW 240.23).
Figure imgf000034_0001
To a mixture of 5-acetamido-2-methoxy-3-methylbenzoquinine (20.2 mg, 0.10 mmol) and 2-fluoroethylamine hydrochloride (100 mg, 0.90 mmol) in ethanol (6 ml) was added sodium hydroxide solution (36 mg, 0.90 mmol, in 0.15 ml water) at room temperature with stirring. Upon the complete conversion of the starting material shown by thin layer chromatography (5 hours), the mixture was poured into water, and extracted with methylene chloride. The organic layer was dried over anhydrous sodium sulfate, and concentrated. Separation by flash column chromatography on silica gel (2:1 hexane/ethyl acetate) afforded the product as a purple solid (20.5 mg, 88%). 1H NMR (CDCl3, 500 MHz) δ 8.58 (s, IH), 7.28 (s, IH), 6.13 (bs, IH), 4.57 (dt, J= 47.0, 5.0 Hz, 2H), 3.87 (ddt, J= 26.0, 5.5, 5.0 Hz, 2H), 2.20 (s, 3H), 2.06 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ 184.1, 180.2, 169.4, 144.9, 140.4, 109.5, 105.1, 82.3 (d, J= 170 Hz), 45.1 (d, J= 20 Hz), 25.1, 10.4. HRMS (FAB) found 241.0987 [M+H]+, calcd. 241.0989 for CnHi4N2O3F. IR (KBr) (cm"1) 3276, 2918, 2850, 1712, 1645, 1581, 1522, 1466, 1348, 1277, 1194. UV-VIS (EtOH) λ 340, 505 nm. 5-Acetamido-2-allylamino-3-methylbenzoquinone ("AAQ", MW 234.25)
Figure imgf000034_0002
To a solution of 5-acetamido-2-methoxy-3-methylbenzoquinine (8.0 mg, 0.04 mmol) in methylene chloride (5.0 ml) was added allylamine (35 μl, 0.47 mmol) at room temperature with stirring. Upon the complete conversion of the starting material shown by thin layer chromatography (24 hours), the mixture was washed with water, dried over anhydrous sodium sulfate, and concentrated. Separation by flash column chromatography on silica gel (3:1 hexane/ethyl acetate) afforded the product as a purple solid (8.0 mg, 89%). 1H NMR (CDCl3, 500 MHz) δ 8.63 (bs, IH), 7.26 (s, IH), 6.17 (bt, J= 6.0 Hz, IH), 5.93-5.85 (m, IH), 5.25-5.19 (m, 2H), 4.15-4.12 (m, IH), 2.20 (s, 3H), 2.05 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ 184.2, 180.0, 169.4, 144.9, 140.7, 134.0, 117.3, 109.2, 104.2, 46.8, 25.0, 10.0. HRMS (FAB) found 235.1084 [M+H]+, calcd. 235.1083 for Ci2Hi5N2O3. IR (KBr) (cm"1) 3284, 2920, 2852, 1707, 1655, 1568, 1516, 1477, 1346, 1288, 1194. UV-VIS (EtOH) λ 340, 520 nm.
5-Acetamido-2-(l-aziridinyl)-3-methylbenzoquinone ("ARQ", MW 220.22)
Figure imgf000035_0001
To a solution of 5-acetamido-2-methoxy-3-methylbenzoquinine (8.3 mg, 0.04 mmol) in methylene chloride (2.5 ml) was added aziridine [Allen, CFH et ah, Org. Synth. Coll., 1963, 4, 433] (0.20 ml, 3.87 mmol) at room temperature with stirring. Upon the complete conversion of the starting material shown by thin layer chromatography (30 min), the mixture was washed with water, dried over anhydrous sodium sulfate, and concentrated. Separation by flash column chromatography on silica gel (2:1 hexane/ethyl acetate) afforded the product as a dark orange solid (8.1 mg, 93% yield) 1H NMR (CDCl3, 500 MHz) δ 8.24 (bs, IH), 7.29 (s, IH), 2.32 (s, 4H), 2.19 (s, 3H), 2.01 (s, 3H). 13C NMR (CDCl3, 75 MHz) δ 183.9, 182.1, 169.2, 152.5, 138.3, 122.3, 112.3, 28.9, 24.9, 9.5. HRMS (FAB) found 221.0925 [M+H]+, calcd. 221.0926 for CnHi3N2O3. IR (KBr) (cm 1) 3325, 2931, 2852, 1714, 1653, 1628, 1593, 1508, 1381, 1352, 1288, 1200, 1165. UV-VIS (EtOH) λ 340, 475 nm.
5-Acetamido-2-(2-acetamidoethyl)amino-3-methylbenzoquinone ("AAEQ") MW 279.29
Figure imgf000035_0002
To a solution of 5-acetamido-2-methoxy-3-methylbenzoquinine (15.0 mg, 0.07 mmol) in methylene chloride (10 ml) was added 2-acetamidoethylamine (92 μl, 0.86 mmol) at room temperature with stirring. [When a greater equivalent amount of 2-acetamidoethylamine was used, an additional product tentatively identified as 6-acetamido-8-methyl-7-oxo-3,7- dihydro-2H-quinoxaline was obtained.] Upon the complete conversion of the starting material shown by thin layer chromatography (8 hours), the mixture was washed with water, dried over anhydrous sodium sulfate, and concentrated. Separation by flash column chromatography on silica gel (ethyl acetate) afforded the product as a purple solid (15.4 mg, 77%). 1H NMR (CDCl3, 500 MHz) δ 8.60 (bs, IH), 7.23 (s, IH), 6.25 (bt, J= 6.0 Hz, IH), 5.80 (bt, J= 6.0 Hz, IH), 3.71 (td, J= 11.0, 6.0 Hz, 2H), 3.46 (td, J= 11.0, 6.0 Hz,
2H), 2.20 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H). HRMS (FAB) found 280.1298 [M+H]+, calcd. 280.1297 for Ci3Hi8N3O4. IR (KBr) (cm"1) 3269, 2918, 2848, 1687, 1655, 1568, 1516, 1483, 1371, 1281, 1196. UV-VIS (EtOH) λ 340, 520 nm. 5-Acetamido-2-(6-acetamidohexyl)amino-3-methyl- benzoquinone ("AAHQ") MW: 335.40
Figure imgf000036_0001
To a solution of 5-acetamido-2-methoxy-3-methylbenzoquinine (9.5 mg, 0.05 mmol) in methylene chloride (3.0 ml) was added 6-acetamidohexylamine (52 mg, 0.33 mmol) at room temperature with stirring. Upon the complete conversion of the starting material shown by thin layer chromatography (6 hours), the mixture was washed with water, dried over anhydrous sodium sulfate, and concentrated. Separation by flash column chromatography on silica gel (20:1 ethyl acetate/methanol) afforded the product as a purple solid (13.1 mg, 86%). 1H NMR (CDCl3, 500 MHz) δ 8.65 (bs, IH), 7.23 (s, IH), 6.04 (bt, J= 6.0 Hz, IH), 5.49 (bs, IH), 3.52 (td, J= 7.0, 6.0 Hz, 2H), 3.21 (td, J= 7.0, 6.0 Hz, 2H), 2.19 (s, 3H), 2.07 (s, 3H), 1.96 (s, 3H), 1.60 (tt, J= 7.0 Hz, 2H), 1.49 (tt, J= 7.0 Hz, 2H), 1.41-1.29 (m, 4H). 13C NMR (CDCl3, 125 MHz) δ 184.3, 179.7, 170.1, 169.5, 145.0, 140.8, 108.9, 103.5, 44.8, 39.3, 30.5, 29.5, 26.4, 26.3, 25.0, 23.3, 10.3. HRMS (FAB) found 336.1925 [M+H]+, calcd. 336.1923 for CnH26N3O4. IR (KBr) (cm 1) 3276, 2931, 2852, 1711, 1647, 1570, 1518, 1477, 1352, 1281, 1194. UV-VIS (EtOH) λ 340, 520 nm. 5-Acetamido-2-(8-acetamido-3,6-dioxaoctyl)- amino-3-methylbenzoquinone ("AATEQ) MW: 367.40"
Figure imgf000037_0001
To a solution of 5-acetamido-2-methoxy-3-methylbenzoquinine (10.1 mg, 0.05 mmol) in methylene chloride (3.0 ml) was added 2,2'-(ethylenedioxy)bis(ethylamine) (0.25 ml, 1.71 mmol) at room temperature with stirring. Upon the complete conversion of the starting material shown by thin layer chromatography (1 hour), the mixture was washed with water, dried over anhydrous potassium carbonate, and stirred with acetic anhydride (0.30 ml) and DMAP (80 mg). After 30 min, the mixture was washed with diluted hydrochloric acid and saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated. Separation by flash column chromatography on silica gel (10:1 ethyl acetate/methanol) afforded the product as a purple solid (16.0 mg, 90%). 1H NMR (CDCl3, 500 MHz) δ 8.64 (bs, IH), 7.18 (s, IH), 6.40 (bs, IH), 6.37 (bs, IH), 3.71 (td, J= 5.0, 5.0 Hz, 2H), 3.62 (t, J= 5.0 Hz, 2H), 3.61-3.55 (m, 4H), 3.51 (t, J= 5.0 Hz, 2H), 3.40 (td, J = 5.0, 5.0 Hz, 2H), 2.16 (s, 3H), 2.02 (s, 3H), 1.92 (s, 3H). 13C NMR (CDCl3, 75 MHz) δ 184.3, 179.6, 170.2, 169.4, 145.1, 140.7, 108.9, 104.1, 70.3, 70.0, 69.9, 69.2, 44.4, 39.3, 24.9, 23.1, 10.3. HRMS (FAB) found 368.1824 [M+H]+, calcd. 368.1822 for CnH26N3O6. IR (KBr) (cm"1) 3276, 2933, 2877, 1709, 1645, 1570, 1518, 1462, 1342, 1275, 1188. UV- VIS (EtOH) λ 340, 510 nm. S-Acetamido^-carboxymethylamino-S-methylbenzoquinone ("CMQ") MW: 252.22
Figure imgf000037_0002
To a mixture of 5-acetamido-2-methoxy-3-methylbenzoquinine (5.5 mg, 0.03 mmol) and glycine (21.2 mg, 0.28 mmol) in ethanol (2 ml) was slowly added sodium hydroxide solution (10 mg, 0.25 mmol, in 0.10 ml water) at room temperature with stirring. The mixture turned dark immediately. Upon the complete conversion of the starting material shown by thin layer chromatography, the mixture was acidified with diluted hydrochloric acid, and extracted with ethyl acetate. Purification by basic-acidic extractions afforded the product as a purple solid (5.9 mg, 89%). 1U NMR (Acetone-d6, 500 MHz) δ 9.01 (bs, IH),
7.20 (s, IH), 6.64 (bs, IH), 4.43 (d, J= 6.0 Hz, 2H), 2.25 (s, 3H), 2.02 (s, 3H). HRMS (FAB) found 253.0823 [M+H]+, calcd. 253.0825 for CnHi3N2O5. IR (KBr) (cm 1) 3311, 2927, 2852, 1736, 1714, 1653, 1587, 1491, 1377, 1196. UV-VIS (EtOH) λ 340, 510 nm.
EXAMPLE III Identification of Targets of GA and Effects on Cells To screen for a novel target(s) of GA, the present inventors used lysates of canine
MDCK cells and human DBTRG glioblastoma cells which are sensitive to certain GA drugs when measured in uPA-plasmin activation assays and in vitro scattering and invasion assays .
Using a modification of the procedure described by Whitesell et al. {supra), GA affinity binding beads were made using a C6-GA bead linker (Fig. 5) for use in "pulldown" experiments to identify potential GA-binding proteins in cell lysates. As determined by LC-MS/MS analysis (Table 1) of SDS PAGE Coomassie-stained protein bands from either MDCK or DBTRG cell lysates, GA-coupled beads pulled down HSP90 (Fig. IA and Table 1) as originally described by Whitesell et al. {supra). In addition, other proteins were pulled-down, including myosin, actin, tubulin, and GAPDH (not shown). However, a 32-kDa protein, an outer membrane mitochondrial protein, VDAC, was identified in both MDCK and DBTRG cell lysates (Fig. IA, Table 1).
Table 1. Peptide MS/MS Results (supporting protein identifications)
Figure imgf000039_0001
Cysteine alkylated during sample preparation
Peptide Mass Tolerance ± 2 Da Fragment Mass Tolerance ± 0.1 Da Max Missed Cleavages 2 Instrument type ESI-OUAD-TOF
When MDCK and DBTRG cells were pre-treated for 24 h with either GA or 17- allylamino-17-demethoxygeldanamycin (17-AAG), both drugs effectively competed for HSP90 binding in the pull-down assay (as observed by Coomassie blue staining of the gel). GA appeared to be a more effective competitor (Fig. IA). Even though both 17-AAG and GA competed for HSP90 binding (Fig. IA, lanes 4 and 5, respectively), VDAC binding was not affected, despite the much greater abundance of HSP90 in the cells.
Testing of other linkers to couple the compound to the beads (Fig. 5) gave results (for HSP90 and VDAC) that
(i) were similar to the C6-GA linker such as with C12-GA and C12-6A (Fig. 6, lane 2) (Linkers shown in Fig. 5),
(ii) showed preference for HSP90 in the pull-down (TEG-GA in Fig. 5; Fig. 6, lane 3), or
(iii) required the ansamycin ring for activity (C6-AQ in Fig. 5; Fig. 6, lane 4). To show that VDAC was precipitated directly from mitochondria and not indirectly, mitochondria purified from either MDCK and DBTRG cells were subjected to GA-bead pulldown assays as above (Fig. IB). Both HSP90 (Fig. IB, upper panel) and VDAC (lower panel) were found in the post-nuclear cell lysate (lane 1), whereas VDAC was exclusively found in the purified mitochondrial fraction (Fig. IB, lane 3) or in the supernatant fraction of the mitochondrial wash (Fig. IB, lane 2). In the post-pull-down supernatant fractions, HSP90 was mainly in the post-nuclear pull-down supernatant whereas only VDAC was detected in the post-mitochondrial GA pull-down supernatant (Fig. 1, lanes 4-6). These results indicated that the mitochondrial pore protein binds directly to GA affinity beads in a manner that is independent of HSP90.
As noted above, VDAC is a component of the mitochondrial PTP, a calcium-, voltage- sensitive, and pH-sensitive pore that opens when calcium levels increase, especially when accompanied by oxidative stress (Green et ah, supra; Marzo et ah, supra). To measure whether GA influences mitochondrial function related to PTP and VDAC, a number of well- known inhibitors of the PTP and VDAC, including 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid, disodium salt (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid, disodium salt (H2DIDS) (D. Han et al, J Biol Chem 278, 5557 (2003)), UbO, and Decyl- Ub were tested for their ability to inhibit HGF/SF-mediated uPA-plasmin activation (Fig. 2). DIDS is a nonspecific, voltage-dependent VDAC inhibitor that prevents O2 diffusion from the intermembrane space to the cytoplasm. At 0.5 mM, DIDS inhibited superoxide production in isolated heart mitochondria (Han et al, supra). Both DIDS (not shown) and H2DIDS at 100 μM inhibited uPA-plasmin activation (Fig. 2). Importantly, both UbO and Decyl-Ub benzoquinones, both of which are believed to bind to VDAC (E. Fontaine et al., J Biol Chem 273, 25734 (1998); AM Cesura et al., J Biol Chem 278, 49812 (2003)), blocked Ca2+ influx into mitochondria by closing the PTP pore. Both molecules also interfere with electron transfer and are involved in ROS production (S. Dikalov et al., J Biol Chem 211 , 25480 (2002). The present results showed that UbO at 10 μM and Decyl-Ub at 100 μM significantly inhibited HGF/SF-induced uPA-plasmin activation (Fig. 2). These are the same concentrations that blocked Ca2+ influx in purified mitochondria (Fontaine et al., supra; S. Martinucci et al, FEBS Lett 480, 89 (2000)), again, with UbO displaying the highest activity. Cyclosporin A (CsA) was also tested; this drug, at 1-5 μM, reduces PTP pore activity by inhibiting the adenine nucleotide translocator (AdNT), preventing ATP and ADP translocation (Fontaine et al, supra). CsA showed some inhibition of uPA activity at 100 μM but not at 10 μM (Fig. 2/3). By targeting VDAC, UbO and Decyl-Ub may block ion transfer through the mitochondrial PTP (Dikalov et al, supra; J. Ou et al, Am J Physiol. 286, H561 (2004)). That they inhibit HGF/SF-induced uPA-plasmin activation and MDCK scattering implies that VDAC function is required to regulate HGF/SF-mediated cell motility.
Benzoquinone moieties of GA derivatives shown below were tested for activity in the MDCK-HGF/SF induced uPA assay. One derivative, 5-Acetomido-2-(l-aziridinyl)-3-methyl- benzoquinone (ARQ) the benzoquinone found in 17-(l-aziridinyl)-17- demethoxygeldanamycin (ARG) displayed nanomolar inhibitory activity (Table 2 and Fig. 3) and, therefore, according to the present invention, has therapeutic activity, based on its inhibitory concentration above, which is two orders of magnitude lower than the inhibitory concentration of UbO and in the same potency range as several of the "active" GA derivatives (the full sized molecules). A summary of the concentration ranges in which various of the GA and derivative compounds and the benzoquinones of the present invention exert their inhibitory action in the uPA assay described herein are shown in Table 2B
UbO, Decyl-Ub, and GA each include a benzoquinone group and are members of a family of "anti-tumor quinones" that are metabolized by NADPH: quinone oxidoreductase 1 (NQOl, DT-diaphorase) (D. Ross, Drug Metab Rev 36, 639 (2004)) and are processed in mitochondria. As stated above, UbO and Decyl-Ub blocked PTP pore activity by regulating Ca2+ permeability, an effect that could be reversed by increasing Ca2+ concentration (Martinucci et al, supra). By contrast, HGF/SF stimulation of cell migration increases Ca2+ influx through a plasma-membrane transient receptor potential cation channel, TRPVl (J.
Vriens et al, Cell Calcium 36, 19 (2004)). Collapsing the mitochondrial membrane potential or inhibiting the Ca2+ importer prevents development of store-operated Ca2+ entry through the plasma membrane as determined by measurement of the Ca2+ release-activated Ca2+ current (ICRAC) (JA Gilabert et al. , EMBO J 19, 6401 (2000) ; JA Gilabert et al, EMBO J 20, 2672 (2001)). This suggests that Ca2+ influx through TRP channels is a key step in HGF/SF- mediated cell motility and a potential site for indirectly monitoring the mitochondria VDAC- PTP response to inhibition by UbO, Decyl-Ub, and GA.
To test this hypothesis, whole-cell voltage clamp measurements (OP Hamill et al, Pflugers Arch 391, 85 (1981)) were performed on MDCK cells. The results are shown in Fig. 4-4H. These studies were conducted to determine if (a) HGF/SF increased membrane current and (b) the benzoquinones inhibited such current activation. Consistent with HGF/SF treatment increasing hepatocyte intracellular [Ca2+] concentration (G. Baffy et al, J Cell Physiol 153, 332 (1992)) and Ca2+ flux in HepG2 cells (Vriens et al, supra), HGF/SF increased whole-cell, cationic current in MDCK cells (Fig. 4A). The inward and outward currents (recorded at -80 and 80 mV, respectively) were plotted versus time (Fig. 4B).
HGF/SF (100 ng/ml) was added where indicated. Inward current increased within minutes after adding HGF/SF, and this increase reversed immediately when N-methyl-D-glucamine (NMDG) was substituted for all external cations (Fig. 4B). Both inward and outward current increased further over an additional ten minutes. The time course for HGF/SF-stimulated current, both the onset and rate of increase, varied among individual cells (cf. Figs. 4B, D, F, and H). Baffy and colleagues {supra) reported similar variability in individual cells for increases in [Ca2+J1 concentration among rat hepatocytes in response to HGF/SF. Nevertheless, the overall time course is similar to the time course of epidermal growth factor (EGF) stimulation of vesicular translocation and insertion of TRP channels and their corresponding increases in [Ca2+J1 concentration (VJ Bezzerides et al., Nat Cell Biol 6, 709 (2004)).
TABLE 2: Inhibition of Met-mediated uPA Activity by Various GA Derivatives and the Benzoquinones ARQ and UbO1
Figure imgf000043_0001
Listed in order of decreasing uPA indices. The two benzoquinone compounds are bolded. *IC50 is the concentration at which 50% inhibition is achieved
**The uPA-plasmin inhibition index is the negative loglO of the drug concentration at which 50% inhibition of uPA occurs (Webb et. al. 2000). Table 2B Activity of GA and Derivatives and Benzoquinone Compounds in uPA Assay
Figure imgf000044_0001
Compounds designated by numbered are are GA and derivatives, Compound designated by letter are benzoquinones (see Figure 7)
An experiment was performed to test whether the VDAC-PTP inhibitors UbO and Decyl-Ub (Fig. 4C-F) influenced cationic currents induced by HGF/SF. Both agents rapidly reversed HGF/SF-stimulated increases of cationic current in MDCK cells (Figs. 4C - 4F; Table 2) at lOμM concentration, which is known to inhibit the PTP (Martinucci et al., supra). GA was next tested and results showed that picomolar concentrations (10" M) inhibited inward and outward currents approximately six minutes after addition (Fig. 4G and 4H).
GA therefore is a highly active inhibitor of Ca2+ flux. Membrane currents plotted as a function of the voltage ramps were taken at the indicated times. HGF/SF increased the slope conductance accompanied by a shift in reversal potential toward 0 mV, whereas subsequent addition of GA (10~12 M) reduced the slope conductance (Table 2).
The fact that benzoquinone drugs all reduce HGF/SF-induced cation current indicates that they have a common target. The fact that the GA displays activity at picomolar levels is significant. A negative control compound, radicicol was tested; this compound (2, 3) has much higher affinity for HSP90 than GA and has activity against uPA at nanomolar concentrations, presumably where it inhibits HSP90 function.
Radicicol at 10 pM had no effect on HGF/SF-stimulated current; however, at 10 nM it markedly inhibited stimulation by HGF/SF (Table 3). A summary of the effects of all agents and conditions on slope conductance is shown in Table 3. HGF/SF stimulates a nonselective cation current in MDCK cells, similar to its effect on HepG2 cells (Vriens et al., supra). It is significant that UbO and Decyl-Ub, known inhibitors of the mitochondrial VDAC and PTP (Fontaine et al., supra), also reduce HGF/SF- activated increases in slope conductance in the same concentration range that inhibited uPA- plasmin activity (cf. Fig. 3 to Fig. 4C-F) and with a time course similar to that of GA- mediated inhibition (Fig. 4H). Moreover, radicicol only inhibited slope conductance at 10 nM concentration (Table 2), but had no effect at picomolar concentrations on either untreated or HGF/SF-treated MDCK cells (Table 2).
Since GA inhibition occurred 6 min following addition to the cells, and considering that it had no direct effect when added to outside-out membrane patches from DBTRG cells that contained active cation channels (not shown), it was concluded that another drug target — namely mitochondrial VDAC — mediates some of the GA inhibition by influencing membrane currents and mitochondria function at nanomolar concentrations. TABLE 3. Inhibition of HGF/SF-Induced Cationic Currents
Figure imgf000046_0001
tMean ± SEM* Differs significantly from HGF/SF treatment, p < 0.05 as determined by Student Newman-Keuls multiple comparison of means
§ Differs significantly from control, p < 0.05. nS = nanoSiemens pF = picoFarads
GA targets HSP90, and, because of HSP90's function as a chaperone, has a broad spectrum of effects, including on Met (Webb et al., supra; Xie et al, supra). Because of this, effects mediated through mitochondria would be masked. Inhibition of cationic currents in "control" cells treated with GA at picomolar levels is shown in Table 2). UbO also has inhibitory activity in control cells not treated with HGF/SF (Table 3). This is further evidence that benzoquinone compounds act as general inhibitors of mitochondrial activity, and can inhibit cell invasion activity at lower concentrations than those affecting HSP90.
UbO also has inhibitory activity in control cells not treated with HGF/SF (Table 1), further supporting the notion that the benzoquinone compounds are general inhibitors of mitochondrial activity that is associated with cell invasion. GA, UbO, and Decyl-Ub, each including benzoquinone, inhibit both HGF/SF-induced membrane current and uPA activity. GA acts at nanomolar concentrations whereas both UbO and Decyl-Ub act at micromolar concentrations (Fig. 3, Table 3). However, the benzoquinone compound, ARQ, acts at nanomolar levels. This is strong evidence that GA has quinone-like bioactivity, as previously indicated (S. Dikalov et al., supra; VG Brunton et al., Cancer Chemother Pharmacol 41, 417 (1998)). In fact, GA uncoupled endothelial nitric-oxide synthase (eNOS) activity and increased endothelial NOS-dependant superoxide production (Dikalov et al, supra; SS Billecke et al., J Biol Chem 277, 20504 (2002)) to a greater degree than did radicicol, which lacks the benzoquinone moiety. Moreover, when eNOS, a substrate of HSP90, was blocked with L- (N(omega)-nitro-L-arginine methyl ester, GA still increased superoxide while radicicol did not (J. Ou et al, Am J Physiol Heart Circ Physiol 286, H561 (2004)), implicating the benzoquinone moiety. These findings suggested that GA and radicicol inhibited NO by different mechanisms. GA uncouples both eNOS and redox cycling while radicicol only uncouples eNOS (Ou et al, supra). GA, like other redox-cycling quinones, directly affects redox cycles with eNOS by a process independent of its action on HSP90 (Dikalov et al, supra; Billecke et al, supra).
"Redox-cycling quinones" can participate in catalytic electron transfer processes via an intermediate one-electron reduced semiquinone form and transition to either the quinone (fully oxidized) or the hydroquinone (fully reduced) state. UbO and Decyl-Ub, both tested by the present inventors, belong to this category. However, GA works at concentrations orders of magnitude lower than UbO and Decyl-Ub, both in inhibiting cell scattering and uPA- plasmin activation as well as in whole-cell voltage clamp assays (Table3).
All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.
Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

Claims

WHAT IS CLAIMED IS:
1. A redox-cy cling benzoquinone, semiquinone or hydroquinone compound, or a pharmaceutically acceptable salt of the benzoquinone , which compound has the property of inhibiting (a) the activation of Met by HGF/SF in cancer cells and (b) HGF/SF-induced tumor cell invasion, which benzoquinone, semiquinone, hydroquinone or compound or salt binds to and inhibits the activity of mitochondrial Voltage Dependant Anion Channel (VDAC) protein of cells.
2. The compound of claim 1, wherein the benzoquinone is present in the structure of geldanamycin (GA) or a 17-alkylamino 17-demethoxy derivative of GA OR FEG.
3. The compound of claim 2 wherein the inhibition of VDAC equals or exceeds the inhibitory action of mitochondrial specific VDAC inhibitors ubiquinone (UbO) or decyl- ubiquinone (Decyl-Ub).
4. The compound of claim 3 that inhibits non-selective cation currents in said cells at subnanomolar concentrations.
5. The compound of claim 4 that inhibits non- selective cation currents in said cells at concentrations of < 10"10M.
6. The compound of claim 3 that inhibits Ca2+ influx, thereby acting as a calcium channel blocker.
7. The compound of claim 1 wherein the inhibition of VDAC equals or exceeds the inhibitory action of mitochondrial specific VDAC inhibitors ubiquinone (UbO) or decyl- ubiquinone (Decyl-Ub).
8. The compound of claim 7 that inhibits non-selective cation currents in said cells at subnanomolar concentrations.
9. The compound of claim 8 that inhibits non- selective cation currents in said cells at concentrations of < 10"10M.
10. The compound of claim 7 that inhibits Ca2+ influx, thereby acting as a calcium channel blocker.
11. The compound of any of claims 1-10 that is selected from the group consisting of: 5-Acetamido-2-methoxy-3-methylbenzoquinone (GQ) , 5-Acetamido-2-(2-fluoroethyl)amino-3-methylbenzoquinone (FEQ) , 5-Acetamido-2-allylamino-3-methylbenzoquinone (AAQ) , 5-Acetamido-2-( 1 -aziridinyl)-3-methylbenzoquinone (ARQ) , 5-Acetamido-2-(2-acetamidoethyl)amino-3-methylbenzoquinone (AAEQ) ,
5-Acetamido-2-(6-acetamidohexyl)amino-3-methylbenzoquinone (AAHQ), 5-Acetamido-2-[2-[2-(2-acetamidoethyloxy)ethoxy]ethyl]amino-3- methylbenzoquinone (AATEQ), and
5-Acetamido-2-carboxymethylamino-3-methylbenzoquinone (CMQ) .
12. The compound of claim 11 that is ARQ.
13. A pharmaceutical compositions comprising: (a) the compound of any of claims 1-10; and (b) a pharmaceutically acceptable carrier or excipient.
14. A pharmaceutical compositions comprising:
(a) the compound of claim 11 ; and
(b) a pharmaceutically acceptable carrier or excipient.
15. A method of inhibiting function of mitochondrial Voltage Dependant Anion Channel (VDAC) protein and thereby inhibiting cation flux in a cell, comprising providing to the cell an effective amount of:
(i) geldanomycin (GA) or a derivative thereof, or (ii) a pharmaceutically acceptable salt of the GA or the derivative, which GA, derivative or salt binds to and inhibits VDAC function and cation flux at subnanomolar concentrations, and which GA or derivative includes in its structure a redox- cy cling benzoquinone moiety.
16. A method of inhibiting a HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell, comprising providing to said cells an effective amount of the compound of any of claims 1-10, or the pharmaceutical composition of claim 13, wherein the compound has an IC50 of less than about 10~9 M for inhibition of said biological activity.
17. The method of claim 16 wherein the compound has an IC50 of less than about 10~8 M for inhibition of said biological activity.
18. The method of claim 16 wherein the compound has an IC50 of less than about 10~7 M for inhibition of said biological activity.
19. A method of inhibiting a HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell, comprising providing to said cells an effective amount of the compound of any of claims 1-10, or the pharmaceutical composition of claim 14, wherein the compound has an IC50 of less than about 10"9 M for inhibition of said biological activity.
20. The method of claim 19 wherein the compound has an IC50 of less than about 10"8 M for inhibition of said biological activity.
21. The method of claim 19 wherein the compound has an IC50 of less than about 10~7 M for inhibition of said biological activity.
22. A method of inhibiting a HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell, comprising providing to said cells an effective amount of the compound of claim 11 , or the pharmaceutical composition of claim 13, wherein the compound has an IC50 of less than about 10~9 M for inhibition of said biological activity.
23. The method of claim 22 wherein the compound has an IC50 of less than about 10~8 M for inhibition of said biological activity.
24. The method of claim 22 wherein the compound has an IC50 of less than about 10~7 M for inhibition of said biological activity.
25. A method of inhibiting a HGF/SF-induced, Met receptor mediated biological activity of a Met-bearing tumor or cancer cell, comprising providing to said cells an effective amount of the compound of claim 11 , or the pharmaceutical composition of claim 14, wherein the compound has an IC50 of less than about 10~9 M for inhibition of said biological activity.
26. The method of claim 25 wherein the compound has an IC50 of less than about 10~8 M for inhibition of said biological activity.
27. The method of claim 25 wherein the compound has an IC50 of less than about 10~7 M for inhibition of said biological activity.
28. The method of claim 16 wherein said biological activity is the induction of uPA activity in said cells.
29. The method of claim 19 wherein said biological activity is the induction of uPA activity in said cells.
30. The method of claim 22 wherein said biological activity is the induction of uPA activity in said cells.
31. The method of claim 25 wherein said biological activity is the induction of uPA activity in said cells.
32. The method of claims 15 wherein said biological activity is growth, scatter or invasion of said cells.
33. The method of claims 16 wherein said biological activity is growth, scatter or invasion of said cells.
34. The method of claims 19 wherein said biological activity is growth, scatter or invasion of said cells.
35. The method of claims 22 wherein said biological activity is growth, scatter or invasion of said cells.
36. The method of claims 25 wherein said biological activity is growth, scatter or invasion of said cells.
37. The method of claim 28 wherein said invasion results in tumor metastasis.
38. The method of claim 29 wherein said invasion results in tumor metastasis.
39. The method of claim 30 wherein said invasion results in tumor metastasis.
40. The method of claim 31 wherein said invasion results in tumor metastasis.
41. A method of inhibiting in a subject metastasis of Met-bearing tumor or cancer cells that is induced by HGF/SF, comprising providing to said subject an effective amount of the compound of any of claims 1-10, or the pharmaceutical composition of claim 13, wherein the compound has an IC50 of less than about 10~9 M for inhibition tumor cell invasion when measured in an assay in vitro.
42. A method of inhibiting in a subject metastasis of Met-bearing tumor or cancer cells that is induced by HGF/SF, comprising providing to said subject an effective amount of the compound of any of claims 1-10, or the pharmaceutical composition of claim 14, wherein the compound has an IC50 of less than about 10~9 M for inhibition tumor cell invasion when measured in an assay in vitro.
43. A method of inhibiting in a subject metastasis of Met-bearing tumor or cancer cells that is induced by HGF/SF, comprising providing to said subject an effective amount of the compound of claim 11, or the pharmaceutical composition of claim 13, wherein the compound has an IC50 of less than about 10~9 M for inhibition tumor cell invasion when measured in an assay in vitro.
44. A method of inhibiting in a subject metastasis of Met-bearing tumor or cancer cells that is induced by HGF/SF, comprising providing to said subject an effective amount of the compound of claim 11, or the pharmaceutical composition of claim 14, wherein the compound has an IC50 of less than about 10~9 M for inhibition tumor cell invasion when measured in an assay in vitro.
45. The method of claim 16 wherein said inhibition results in measurable regression of a tumor caused by said cells or measurable attenuation of tumor growth in said subject.
46. The method of claim 19 wherein said inhibition results in measurable regression of a tumor caused by said cells or measurable attenuation of tumor growth in said subject.
47. The method of claim 22 wherein said inhibition results in measurable regression of a tumor caused by said cells or measurable attenuation of tumor growth in said subject.
48. The method of claim 25 wherein said inhibition results in measurable regression of a tumor caused by said cells or measurable attenuation of tumor growth in said subject.
49. The method of claim 41 wherein said inhibition results in measurable regression of a tumor caused by said cells or measurable attenuation of tumor growth in said subject.
50. The method of claim 42 wherein said inhibition results in measurable regression of a tumor caused by said cells or measurable attenuation of tumor growth in said subject.
51. The method of claim 43 wherein said inhibition results in measurable regression of a tumor caused by said cells or measurable attenuation of tumor growth in said subject.
52. The method of claim 44 wherein said inhibition results in measurable regression of a tumor caused by said cells or measurable attenuation of tumor growth in said subject.
53. A method for blocking calcium channels or cation flux in a cell comprising providing to the cells an effective amount of a compound or pharmaceutical composition thereof, which inhibits the activity of the mitochondrial VDAC protein which comprises providing to the cell an effective amount of a compound that is (i) geldanomycin (GA) or (ii) a derivative thereof, or (iii) a redox-cy cling benzoquinone, semiquinone or /hydroquinone moiety of said GA or derivative, or (iv) a pharmaceutically acceptable salt of the GA, derivative or moiety, or a pharmaceutical composition that comprises any of (i) - (iv), which compound binds to and inhibits VDAC function and cation flux at subnanomolar concentrations.
54. The method of claim 53, wherein the compound is a compound according to any of claims 1-10.
55. The method of claim 53, wherein the compound is a compound according to claim 11.
56. A method of for blocking calcium channels or cation flux in a subject, and thereby preventing or treating a disease or a condition that is preventable or treatable by such calcium channel or cation flux blockade, comprising administering to a subject in need thereof a VDAC-inhibitory effective amount of a compound that is (i) geldanomycin (GA) or (ii) a derivative thereof, or (iii) a redox-cycling benzoquinone/semiquinone/hydroquinone moiety of said GA or derivative, or (iv) a pharmaceutically acceptable salt of the GA, derivative or moiety, or a pharmaceutical composition that comprises any of (i) - (iv), which compound binds to and inhibits VDAC function and cation flux at subnanomolar concentrations.
57. The method of claim 56, wherein the compound is a compound according to any of claims 3-10.
58. The method of claim 56, wherein the compound is a compound according to claim 11.
59. The method of any of claim 53 wherein the cell is a human cell or the subject is a human.
60. The method of any of claim 54 wherein the cell is a human cell or the subject is a human.
61. The method of any of claim 55 wherein the cell is a human cell or the subject is a human.
62. The method of any of claim 56 wherein the cell is a human cell or the subject is a human.
63. The method of any of claim 57 wherein the cell is a human cell or the subject is a human.
64. The method of any of claim 58 wherein the cell is a human cell or the subject is a human.
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