WO2003105766A2 - Rsk inhibitors and therapeutic uses thereof - Google Patents
Rsk inhibitors and therapeutic uses thereof Download PDFInfo
- Publication number
- WO2003105766A2 WO2003105766A2 PCT/US2003/018734 US0318734W WO03105766A2 WO 2003105766 A2 WO2003105766 A2 WO 2003105766A2 US 0318734 W US0318734 W US 0318734W WO 03105766 A2 WO03105766 A2 WO 03105766A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- rsk
- group
- hydroxy
- activity
- composition
- Prior art date
Links
- 0 CC(C(*)C(*)C1*)OC1OC1=C(c(cc2*)ccc2O)Oc2cc(O)cc(O)c2C1=O Chemical compound CC(C(*)C(*)C1*)OC1OC1=C(c(cc2*)ccc2O)Oc2cc(O)cc(O)c2C1=O 0.000 description 7
- ZXQWXUBBVZJSMI-FIRPJDEBSA-N C[C@@H]([C@@H]([C@H](C1)OC(C)(C)O)OCc2ccccc2)O[C@H]1Sc1ccccc1 Chemical compound C[C@@H]([C@@H]([C@H](C1)OC(C)(C)O)OCc2ccccc2)O[C@H]1Sc1ccccc1 ZXQWXUBBVZJSMI-FIRPJDEBSA-N 0.000 description 1
- YBOVKFRFMWFKCC-CFVLRQLYSA-N C[C@@H]([C@@H]([C@H]([C@H]1O)O)O)O[C@H]1Sc1ccccc1 Chemical compound C[C@@H]([C@@H]([C@H]([C@H]1O)O)O)O[C@H]1Sc1ccccc1 YBOVKFRFMWFKCC-CFVLRQLYSA-N 0.000 description 1
- SOCNRTCFYKWBFL-UISSTJQFSA-N C[C@@H]([C@@H]([C@H]([C@H]1O)[O]#C)O)OC1SO Chemical compound C[C@@H]([C@@H]([C@H]([C@H]1O)[O]#C)O)OC1SO SOCNRTCFYKWBFL-UISSTJQFSA-N 0.000 description 1
- QZQMGQQOGJIDKJ-IKOZNORXSA-N C[C@@H]([C@@H]([C@H]([C@H]1OC(C)=O)OC(C)=O)OC(C)=O)OC1OC(C)=O Chemical compound C[C@@H]([C@@H]([C@H]([C@H]1OC(C)=O)OC(C)=O)OC(C)=O)OC1OC(C)=O QZQMGQQOGJIDKJ-IKOZNORXSA-N 0.000 description 1
- YPJGKQRQWRZEKO-VVRBALCJSA-N C[C@@H]([C@@H]([C@H]([C@H]1OC(C)=O)OC(C)=O)OC(C)=O)O[C@H]1Sc1ccccc1 Chemical compound C[C@@H]([C@@H]([C@H]([C@H]1OC(C)=O)OC(C)=O)OC(C)=O)O[C@H]1Sc1ccccc1 YPJGKQRQWRZEKO-VVRBALCJSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
Definitions
- the Mitogen-activated Protein Kinase (MAPK) signaling pathway is one key pathway that transduces a large variety of external signals, leading to cellular responses that include growth, differentiation, inflammation and apoptosis. Accordingly, MAPK is activated by several diverse signals under normal conditions. However, improper regulation of MAPK, including hyperactivity, has been associated with many diseased states. More particularly, improper regulation of the Mitogen-activated Protein Kinase (MAPK) pathway is a distinguishing characteristic in many tumors as well as neurological diseased states such as epilepsy.
- Rsk Ribosqmal S6 Kinase
- MAPK Mitogen-activated Protein Kinase
- the paucity of data concerning key biological roles of the Ser/Thr protein kinase Rsk family in somatic cells results primarily from the difficulty in distinguishing Rsk function from those of MAPK itself and of the many other downstream MAPK effectors. This difficulty has arisen because of the lack of any Rsk-specific inhibitors. Accordingly, a Rsk specific inhibitor is highly desirable for use as a tool for investigating Rsk function under normal conditions and under diseased conditions in which regulation of the MAPK signaling pathway has been compromised.
- the present invention provides a method for screening and identifying Rsk-specific inhibitors, as well as methods for using compositions comprising such inhibitors for the treatment of diseases associated with elevated Rsk activity.
- a composition that comprises a Rsk specific inhibitory compound.
- the composition comprises natural compounds isolated from the plant Forsteronia refracta, or other natural sources, as well as chemically synthesized related compounds that exhibit activity as Rsk specific inhibitors. Inhibition of Rsk by the present compounds has been discovered to halt the proliferation of cancer cell lines while having little effect on the proliferation rate of normal cells. Therefore, the present invention identifies Rsk as a target for therapeutic intervention in diseased states in which the disease or the symptoms can be ameliorated by inhibition of Rsk catalytic activity or Rsk expression. In another embodiment, overexpression of Rsk is used as a diagnostic marker of cancer in individuals.
- Fig. 1 Molecular structure of SLOlOl-1, SL0101-2 and SL0101-3
- Fig. 2 Inhibitory potency of SLOlOl-l, SL0101-2 and SL0101-3.
- the catalytic activity of Rsk in the presence of increasing concentrations of each compound was measured.
- the IC 50 of each compound was determined to be 90nM for SLOlOl-l, 580nM for SL0101-2 and 190nM for SL0101-3.
- Fig. 4 SLOlOl-l inhibits proliferation of transformed cells but not parental cells.
- SLOlOl-1 and kaempferol were measured using kinase assays with an immobilized substrate in the presence of varying concentrations of SL0101-1 or kaempferol.
- the extent of phosphorylation was determined using phosphospecific antibodies directly labeled with horseradish peroxidase (HRP)-conjugated or phosphospecific antibodies in combination with HRP-conjugated secondary antibodies. All assays measured the initial reaction velocity.
- Figs. 6A & 6B SLOlOl-l inhibits activity of the amino-terminal kinase domain.
- Fig. 6A HA-tagged Rsk2 and an HA-tagged truncation mutant containing the Rsk amino-terminal kinase domain (Rsk2 (1-389)) were transfected into baby hamster kidney 21 (BHK21) cells. The HA-tagged proteins were immunoprecipitated from lysates of EGF-stimulated cells.
- Fig. 6A HA-tagged Rsk2 and an HA-tagged truncation mutant containing the Rsk amino-terminal kinase domain (Rsk2 (1-389)) were transfected into baby hamster kidney 21 (BHK21) cells. The HA-tagged proteins were immunoprecipitated from lysates of EGF-stimulated cells.
- HA-tagged proteins including the Rsk2-AIL mutant, wherein the Rsk2 adenosine interacting loop is substituted with that of p70 S6K
- Assays were performed as described in Fig. 5 in the presence of vehicle, 2 ⁇ M SL0101-1 or 2 ⁇ M Ro 318220( a non-specific PKC inhibitor).
- Figs. 7A & 7B SL0101-1 inhibition of cell proliferation is reversible.
- Fig. 7 A Ha-Ras-transformed cells were treated with vehicle or 50 ⁇ M SLO 101-1. After 48 hr the medium was replaced and cells previously incubated with vehicle were maintained in vehicle . Cells that had previously been incubated with SLO 101 - 1 were treated with either SL0101-1 or vehicle (washout). Cell viability was measured 48 hr later.
- Fig. 7B Determination of siRNA to inhibit cancer cell proliferation. Duplex siRNAs to a sequence in the bluescript plasmid (Control), Rskl, Rsk2 or Rskl and Rsk2 were transfected into MCF-7 cells. Medium was replaced 24 hr post-transfection and the cells incubated for an additional 48 hr prior to measuring cell viability.
- Figs. 8A-8D SLO 101-1 inhibits the proliferation of cancer cells but not normal cells.
- Fig. 8 A demonstrates the results of treating MCF-7 and MCF-10A cells with vehicle or 50 ⁇ M SL0101-1 or U0126 (a MEK inhibitor).
- Fig. 8B demonstrates the results of treating LNCaP cells with vehicle or 50 ⁇ M SL0101-1 or 50 ⁇ M U0126.
- Fig. 8C demonstrates the results of treating MCF-7 cells with vehicle or 50 ⁇ M SLOlOl-l in serum-free medium.
- Fig. 8D is a Western blot that presents data showing that SLO 101-1 does not inhibit kinases of the MAPK pathway upstream of Rsk. Cell viability was measured at indicated time points.
- Fig. 9A & 9B specifically activates ER ⁇ - and AR-mediated transcription.
- MCF-7 or LNCaP cells were co-transfected with a luciferase reporter and ⁇ -galactosidase expression vectors. Additionally, the cells were transfected with either control vector (V) or a vector encoding constitutively active Rsk2 (Rsk2(Y707A)).
- the cells were treated with either vehicle, 10 nM estradiol or 5 nM R1881 and/or 100 ng/ml EGF. Luciferase and ⁇ - galactosidase activity were determined and the luciferase data were divided by the ⁇ - galactosidase activity to control for differences in transfection efficiency. The data were normalized so that, in the vector control, the response to vehicle addition was zero and the response to either estradiol or R18181 was 100. The values are +SEM. *P ⁇ 0.05 and **P ⁇ 0.01 (Student's t-test) obtained by comparing the response obtained with the vector control with that obtained with Rsk2 (Y707A).
- Purified SLO 101-1 specifically inhibits Rsk2 activity in vitro. Vehicle or inhibitor (5 ⁇ M) was added to the kinase mix containing 5 nM of the indicated purified kinases. The reaction was allowed to proceed for 30 mins at room temperature and the data were normalized to the kinase activity obtained in the presence of vehicle.
- purified and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment.
- purified does not necessarily indicate that complete purity of the particular molecule has been achieved during the process.
- a “highly purified” compound as used herein refers to a compound that is greater than 90% pure.
- the term "pharmaceutically acceptable canier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
- an "effective amount” means an amount sufficient to produce a selected effect. For example, an effective amount of an Rsk inhibitor is an amount of the inhibitor sufficient to suppress Rsk activity.
- Suppression of Rsk activity can be detected through the use of a serine/threonine kinase assay, such as the kinase assay described in Example 3.
- a serine/threonine kinase assay such as the kinase assay described in Example 3.
- the terms "complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the Watson & Crick base-pairing mles, i.e. two nucleic acid sequences that are capable of binding to one another in an anti-parallel base paring anangement.
- the sequence 5' A-G-T 3' is complementary to the sequence 3' T-C-A 5'.
- Complementarity may be "partial,” in which some of the nucleic acids' bases are not matched according to the base pairing rales. Or, there may be “complete” or “total” complementarity between the nu
- alkyl by itself or as part of another substituent means a straight or branched aliphatic chain having the stated number of carbon atoms.
- halo includes bromo, chloro, fluoro, and iodo.
- haloalkyl refers to an alkyl radical bearing at least one halogen substituent, for example, chloromethyl, fluoroethyl or trifluoromethyl and the like.
- Ci-C n alkyl wherein n is an integer, as used herein, refers to a branched or linear alkyl group having from one to the specified number of carbon atoms.
- C ⁇ -C 6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
- C 2 -C n alkenyl wherein n is an integer, as used herein, represents an olefinically unsaturated branched or linear group having from 2 to the specified number of carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, 1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl, and the like.
- C 2 -C n alkynyl wherein n is an integer, refers to an unsaturated branched or linear group having from 2 to the specified number of carbon atoms and at least one triple bond.
- C ⁇ -C 4 alkoxy represents a group of the structure
- aryl refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.
- Aryl groups can be unsubstituted or substituted with one, two or three substituents independently selected from lower alkyl, haloalkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxy, halo, and nitro.
- Substituted aryl includes aryl compounds having one or two C ⁇ -C 6 alkyl, halo or amino substituents.
- alkyl refers to any aryl group which is attached to the parent moiety via the alkyl group.
- heterocyclic group refers to a C3-C8 cycloalkyl group containing from one to three heteroatoms wherein the heteroatoms are selected from the group consisting of oxygen, sulfur, and nitrogen.
- bicyclic represents either an unsaturated or saturated stable 7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclic ring may be attached at any carbon atom which affords a stable structure.
- the term includes, but is not limited to, naphthyl, dicyclohexyl, dicyclohexenyl, and the like.
- lower alkyl refers to branched or straight chain alkyl groups comprising one to eight carbon atoms, including methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl and the like.
- parenteral means not through the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous.
- treating includes administering therapy to prevent, cure, or alleviate/prevent the symptoms associated with, a specific disorder, disease, injury or condition.
- treating cancer includes inhibition or complete growth anest of a tumor, reduction in the number of tumor cells, reduction in tumor size, inhibition of tumor cell infiltration into peripheral organs/tissues, inhibition of metastasis as well as relief, to some extent, of one or more symptoms associated with the disorder.
- the treatment of cancer also includes the administration of a therapeutic agent that directly decreases the pathology of tumor cells, or renders the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
- neoplastic cells as used herein relates to cells that constitute an abnormal new growth, i.e.
- tumor refers to a mass or population of cells that result from excessive cell division and serve no physiological function in the host organism, whether malignant or benign.
- a “tumor” is further defined as two or more neoplastic cells.
- Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cellular proliferation, they will invade sunounding tissues and may additionally metastasize.
- neoplastic disease relates to any disease that is characterized by the presence of neoplastic cells. Neoplastic diseases include cancer and other diseases characterized by the uncontrolled, abnormal growth of cells.
- cancer examples include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, brain cancer, ovarian cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma.
- anti-tumor agent relates to agents known in the art that have been demonstrated to have utility for treating neoplastic disease.
- antitumor agents include, but are not limited to, antibodies, toxins, chemotherapeutics, enzymes, cytokines, radionuclides, photodynamic agents, and angiogenesis inhibitors.
- Toxins include ricin A chain, mutant Pseudomonas exotoxins, diphtheria toxoid, streptonigrin, boamycin, saporin, gelonin, and pokeweed antiviral protein.
- Chemotherapeutics include 5-fiuorouracil (5-FU), daunorubicin, cisplatinum, bleomycin, melphalan, taxol, tamoxifen, mitomycin-C, and methotrexate as well as any of the compounds described in US Patent No. 6,372,719 (the disclosure of which is inco ⁇ orated herein by reference) as being chemotherapeutic agents,.
- Radionuclides include radiometals.
- Photodynamic agents include po ⁇ hyrins and their derivatives.
- Angiogenesis inhibitors include natural and synthetic biomolecules such as paclitaxel, O-(chloroacetyl-carbomyl) fumagillol ("TNP-470” or “AGM 1470”), thrombospondin-1, thrombospondin-2, angiostatin, human chondrocyte- derived inhibitor of angiogenesis (“hCHIAMP”), cartilage-derived angiogenic inhibitor, platelet factor-4, gro-beta, human interferon-inducible protein 10 ("IP10"), interleukin 12, Ro 318220, tricyclodecan-9-yl xanthate (“D609”), irsogladine, 8,9- dihydroxy-7-methyl- benzo[b]quinolizinium bromide (“GPA 1734”), medroxyprogesterone, a combination of heparin and cortisone, glucosidase inhibitors, genistein, thalidomide, diamino
- Rsk is intended to refer genetically to all the human Rsk isotypes, including Rskl, Rsk2, Rsk3 and Rsk4.
- Rskl, Rsk2, Rsk3 and Rsk4 are specific human isotypes that have previously been described in the literature.
- the nucleic acid and protein sequences of these isotypes are found at Genbank accession numbers NM_002953 (for Rskl, SEQ ID NO: 48), NM_004586 (for Rsk2, SEQ ID NO: 49), NM_021135 (for Rsk3; SEQ ID NO: 50) and NM_014496 (for Rsk4; SEQ ID NO:
- Rsk specific inhibitor includes any compound or condition that inhibits Rsk kinase activity (including any or all of the individual Rsk isotypes) without substantially impacting the activity of other kinases. Such inhibitory effects may result from directly or indirectly interfering with the protein's ability to phosphorylate its substrate, or may result from inhibiting the expression (transcription and/or translation) of Rsk.
- flavonoid refers to polyphenolic compounds possessing a carbon skeleton having the general structure:
- SL0101 is used to refer to the three individual compounds, SLOlOl-l, SL0101-2 and SL0101-3 collectively.
- extract refers to a process of separating and/or purifying one or more components from their natural source, or when used as a noun, refers to the composition produced by such a process.
- the term "antisense oligonucleotide” refers to RNA sequences, as well as the DNA sequences encoding for such RNAs, that are complementary to the sequence of a target RNA (or fragment thereof).
- the target RNA is a mRNA expressed by a cell.
- the term "interfering oligonucleotide” relates to RNA sequences, as well as the DNA sequences encoding for such RNAs, that are capable of inhibiting the function of a target gene product. More particularly, the interfering oligonucleotide is a polynucleotide sequence that comprises a sequence identical or homologous to a target gene (or fragment thereof).
- RNAi interference RNA
- short interfering RNA short interfering RNA
- shRNA short hai ⁇ in RNA
- Short interfering RNAs typically consist of 19-22nt double-stranded RNA molecules that can be chemically synthesized, or generated from larger (>100 nucleotide) double stranded RNA (dsRNA) by enzymatic cleavage using an RNase Ill-like enzyme called Dicer.
- Short hai ⁇ in RNA consists of 19-29nt palindromic sequences connected by loop sequences, that are prepared by chemical synthesis or through recombinant DNA techniques.
- the present invention is directed to compositions comprising a Rsk specific inhibitor and methods of using such compositions for treating disease states related to Rsk hyperactivity.
- Rsk-specific inhibitory activity was first identified in a botanical extract through the use of a novel high throughput screening (HTS) Enzyme-Linked Immunosorbent Assay (ELISA) that produces luminescence as a measure of substrate phosphorylation.
- HTS high throughput screening
- ELISA Enzyme-Linked Immunosorbent Assay
- a dual screen of the extracts was performed using either a constitutively active mutant of isoform 2 of Rsk (Rsk2) or the catalytic domain of the tyrosine kinase, Focal Adhesion Kinase (FAK).
- Forsteronia refracta is a member of the Dogbane family and is native to the South American rain forest. More particularly, the plant is native to Southeastern Brazil, from Goias and Minas Gerais south to Rio Grande do Sul, and in neighboring Misiones, Argentina and Paraguay and is found mostly in upland and riverine forests. Further characteristics of the plant and its availability can be found at http://scisun.nvbg.org:8890/searchdb/owa/wwwspecimen.searchfonn.
- Forsteronia refracta extract contained a general Ser/Thr kinase inhibitor
- activities of the archetypal Ser/Thr kinase, protein kinase A (PKA) and of two kinases most closely related to Rsk2, p70 S6K and Mskl were measured in the presence of varying amounts of extract (Fig. 3).
- Amounts of extract that inhibited Rsk2 activity by 90% did not inhibit PKA, p70 S6K or Mskl to a greater extent than FAK.
- the F. refracta extract contains an inhibitor with remarkable specificity for Rsk2 relative to these other AGC kinase family members.
- a composition comprising an extract from the plant Forsteronia refracta (a member of the dogbane family found in the South American rain forest) wherein the extract has activity as a Rsk specific inhibitor.
- the wood stem and/or stem bark of Forsteronia refracta are extracted with an aqueous solvent to purify flavonoid compounds that have Rsk specific inhibitory activity.
- the present invention is directed to a composition comprising an alcohol (e.g. methanol) extract of wood stem and/or stem bark of Forsteronia refracta, or a derivative product thereof, that contains Rsk specific inhibitory activity.
- the original alcohol extract can be dried to form a powder, or dried and resuspended, or otherwise reconstituted to prepare a non-alcohol solvent based extract comprising the Rsk specific inhibitory compounds of the present invention.
- the present invention is directed to an extract of Forsteronia refracta tissues, wherein the extract comprises one or more of the flavonoids shown in Fig. 1.
- the extract is enriched, relative to other components present in the natural tissues, for flavonoid compounds having Rsk specific inhibitory activity. In other words the flavonoid compounds are present in a higher concentration in the extract relative to their concentration in the natural tissues.
- the extract represents a composition comprising purified flavonoid compounds of E. refracta.
- these three inhibitors do not inhibit the evolutionarily related p70 S6 kinase and Mitogen and Stress-activated Protein Kinase (MSK). In addition, they do not inhibit the prototypical serine/threonine kinase Protein Kinase A or the tyrosine kinase Focal Adhesion Kinase (FAK) (Fig. 3).
- the present invention is directed to compounds represented by the general structure:
- Rj, R 2j and R 3 are independently selected from the group consisting of hydroxy -OCORi, -CORi, C ⁇ -C 4 alkoxy, -O-glucoside and -O-rhamnoside
- R 5 , Re, R 7 , R 8 and R 9 are independently selected from the group consisting of H, hydroxy -OCORi, -CORi, C ⁇ -C 4 alkoxy, -O-glucoside and -O-rhamnoside
- Ri is H or C ⁇ -C alkyl, with the proviso that Ri, R 2 and R 3 are not all hydroxy.
- One embodiment of the invention is directed to a compound of Formula I, wherein R ⁇ , R 2 and R 3 are independently selected from the group consisting of hydroxy and -OCOR t , R 5 and R 9 are each H, R 6, R 7 , and R 8 are independently selected from the group consisting of H, -ORi, -OCOR t , and -COR t and R is H or methyl, with the proviso that Ri, R 2 and R 3 are not all hydroxy.
- Ri and R 2 are independently selected from the group consisting of hydroxy, -CO i, C1-C4 alkoxy and -OCOCH 3 , R 3 is -OCOCH 3 , R t is H or methyl, R 5 , R 8 and R 9 are each H, R 6 is H or hydroxy, and R 7 is hydroxy.
- Ri and R 2 are independently selected from the group consisting of hydroxy and -OCOCH 3 , R 3 is -OCOCH 3 , R 5 , R 8 and R are each H, R 6 is H or hydroxy, and R is hydroxy.
- a compound is provided represented by the general structure of Formula I wherein R ls R 2 and R 3 are independently selected from the group consisting of hydroxy -OC0R 4 , -COR 4 , C ⁇ -C 4 alkoxy, -O-glucoside and -O-rhamnoside, R 4 is H or d-C 4 alkyl, R 5 , R 8 and R 9 are independently selected from the group consisting of H, hydroxy -OCOR 4 , -CCRi and C ⁇ -C 4 alkoxy and Re and R 7 are independently selected from the group consisting of hydroxy -OCOR , -CORi and C1-C4 alkoxy.
- a compound of Formula I wherein Rj, R 2 and R 3 are independently selected from the group consisting of hydroxy and -OCOR , R4 is H or methyl, R 5 and R 9 are each H, R 6 and R 7 are independently selected from the group consisting of hydroxy -OCOR , -COR* and C1-C4 alkoxy, and R 8 is selected from the group consisting of H, -OR t , -OCOR , and -CORi and C ⁇ -C alkoxy.
- a compound of Formula I is provided wherin Ri and R 2 are independently selected from the group consisting of hydroxy and -OCOCH 3 , R 3 is -OCOCH 3 , R5, R 8 and R 9 are each H and R 6 and R 7 are both hydroxy
- R is H or OH
- R ls R 2 and R 3 are independently selected from the group consisting of hydroxy -OCORi, -COR t , C ⁇ -C 4 alkoxy, -O-glucoside and -O- rhamnoside
- Ri is H or -CH 3 , with the proviso that Ri, R 2 and R 3 are not all hydroxy.
- R is H or OH and R] and R 2 are independently selected from the group consisting of hydroxy and -OCOCH 3 and R 3 is -OCOCH 3 .
- R is H and R l9 R 2 and R 3 are independently selected from the group consisting of hydroxy -OCOCH 3 , -COCH 3 , C ⁇ -C 4 alkoxy, -O-glucoside and -O-rhamnoside.
- R is H and Ri
- R 2 and R 3 are independently selected from the group consisting of hydroxy and -OCOCH 3 .
- the compound has the general structure of Formula II wherein R is H, Ri and R 2 are independently selected from the group consisting of hydroxy and -OCOCH 3 and R 3 is -OCOCH 3 . More particularly, in one embodiment a composition is provided comprising one or more compounds having the general structure of Formula II wherein R is H, Ri is hydroxy and R 2 and R 3 are each O O O
- the purified flavonoid compounds and Rsk specific inhibitory extracts of the present invention can be combined with pharmaceutically acceptable carriers, stabilizing agents, solubilizing agents, and fillers known to those skilled in the art to prepare pharmaceuticals for administration to warm blooded vertebrates.
- the compositions can be formulated using standard delivery vehicles and standard formulations for oral, parenteral, inhalation or transdermal delivery.
- Such pharmaceuticals have use in treating neoplastic disease, neurological diseased states (such as epilepsy) or other disease states characterized by inappropriate Rsk activity.
- a Rsk specific inhibitory compound or composition i.e. a Rsk specific inhibitory extract, or specific flavonoid compound
- one or more antitumor agents including those selected from the group consisting of antibodies, toxins, chemotherapeutics, enzymes, cytokines, radionuclides, photodynamic agents, and angiogenesis inhibitors to prepare a pharmaceutical composition.
- Toxins include ricin A chain, mutant Pseudomonas exotoxins, diphtheria toxoid, streptonigrin, boamycin, saporin, gelonin, and pokeweed antiviral protein.
- Chemotherapeutics include 5-fluorouracil (5-FU), daunorubicin, cisplatinum, bleomycin, rnelphalan, taxol, tamoxifen, mitomycin-C, and methotrexate.
- Radionuclides include radiometals.
- Photodynamic agents include po ⁇ hyrins and their derivatives.
- Angiogenesis inhibitors include natural and synthetic biomolecules such as paclitaxel, O-(chloroacetyl-carbomyT) fumagillol ("TNP- 470" or “AGM 1470”), thrombospondin-1, thrombospondin-2, angiostatin, human chondrocyte-derived inhibitor of angiogenesis (“hCHIAMP”), cartilage-derived angiogenic inhibitor, platelet factor-4, gro-beta, human interferon-inducible protein 10 ("IP10"), interleukin 12, Ro 318220, tricyclodecan-9-yl xanthate (“D609”), irsogladine, 8,9- dihydroxy-7-methyl-benzo[b]quinolizinium bromide ("GPA 1734”), medroxyprogesterone, a combination of heparin and cortisone, glucosidase inhibitors, genistein, thalidomide, diamino
- the Rsk specific inhibitor comprises an extract from Forsteronia refracta.ox from the rhizomes of Zingiber zerumbet.
- the Rsk specific inhibitor is an interference RNA or a compound of the general structure of Formula I.
- a composition is provided comprising a chemotherapeutic agent and a compound represented by the general formula
- composition comprises a chemotherapeutic agent and the compound of Formula II or III wherein R is H or OH, Ri and R 2 are independently selected from the group consisting of hydroxy and -OCOCH 3 and R 3 is -OCOCH 3 .
- One aspect of the present invention is directed to a method of preparing a E. refracta extract that exhibits Rsk specific inhibitory activity, wherein the extract is prepared by extracting tissues, selected from the group consisting of wood stem and stem bark of Forsteronia refracta, with an alcohol solution.
- the wood stem and/or bark of E. refracta is contacted with an alcohol (such as methanol), or an alcohol (methanol) containing solution, for a predetermined length of time at room temperature (about 20° to 25° C) with or without agitation.
- the length of time for soaking the plant material can be varied; the tissue simply should be soaked long enough to extract the organic materials in the sample.
- the tissue can be chopped, shredded, ground or macerated/crashed prior to being treated with an aqueous solvent.
- the tissue is sequentially soaked in separate fresh solutions of methanol at room temperature, followed by combination of the methanol solutions and concentration of the solution under diminished pressure to afford a crude extract.
- the tissue is sequentially soaked in three separate fresh solutions of methanol and the methanol solutions are subsequently combined and concentrated.
- the extracted compounds can then be purified using standard techniques.
- the crude extract material can be applied to a polyamide 6S column (such as a 40-g polyamide 6S column) and washed successively with H 2 0, 1 : 1 H 2 O-MeOH, 9: 1 CH 2 Cl 2 -MeOH, 1:1 CH 2 Cl 2 -MeOH and 9:1 MeOH-NH 4 OH to afford five fractions.
- the volume of the washes can be varied, and in one embodiment the column is washed successively with 150 mL of each solvent.
- the 1:1 CH 2 Cl 2 -MeOH fraction is then recovered and potentially further fractionated on a diol gel column (such as a 30-g diol gel column).
- Extracts of the rhizomes of Zingiber zerumbet can be prepared using techniques described in the prior art. For example, fresh rhizomes of Zingiber zerumbet are crashed and extracted (three to four times) with Me 2 CO at room temperature. After filtration, the Me 2 CO is evaporated. The residual H 2 O solution is then extracted with hexane, CH 2 C1 2 and EtOAc, successively.
- the EtOAc layer is concentrated and subjected to Sephadex LH-20 CC, silica gel CC (10-20% MeOH in CHC1 3 , 1% HOAc in EtOAc) and HPLC (column: Develosil ODS-10/20, solv.: 60% MeOH in H 2 O, flow rate: 10 ml min " ) to provide an extract comprising a Rsk inhibitory compound.
- a composition comprising a Rsk inhibitor is prepared from Forsteronia refracta by extracting the tissues as described above.
- the wood stem and/or stem bark of Forsteronia refracta is extracted with an alcohol solution, such as methanol and the extracted material is applied to a polyamide 6S column.
- the 6S column is washed successively with H 2 O, 1:1 H 2 O- MeOH, 9:1 CH 2 Cl 2 -MeOH and 1:1 CH 2 Cl 2 -MeOH to afford four separate fractions, and the 1:1 CH 2 Cl 2 -MeOH fraction is recovered.
- This fraction is then applied to a diol gel column and washed successively with CH 2 C1 2 , 99:1 CH 2 Cl 2 -MeOH and 95:5 CH 2 Cl 2 -MeOH, 90:10 CH 2 Cl 2 -MeOH and MeOH.
- the 95:5 CH 2 Cl 2 -MeOH fraction and the 90: 10 CH 2 Cl 2 -MeOH fraction are recovered as the fractions comprising Rsk inhibitory activity.
- Each of these fractions can be subjected to further purification steps such as a C 18 reverse phase HPLC column with elution using 65:35 MeOH-H 2 O or similar solvent.
- the Rsk-specific inhibitors of the present invention have been shown to inhibit proliferation of a transformed cell without substantially altering the proliferation rate of non-transformed cell growth. Therefore, the inhibitors of the present invention are not toxic to non-transformed cells.
- the specific inhibition of Rsk inhibits proliferation of Ha-ras-transformed NIH/3T3 cells without influencing the proliferation rate of non-transformed NIH73T3 cells.
- Ha-ras-transformed NIH73T3 cells or parental NIH/3T3 cells were incubated in the presence of vehicle, 50 ⁇ M SLO 101-1, or 50 ⁇ M PD 98059, a MEK-specific inhibitor.
- SL0101- 1 inhibits Ha-ras-transformed NIH/3T3 cell proliferation over a 48 hour time course, even in the presence of 10% fetal calf serum (Fig. 4).
- SLO 101-1 had little influence on the rate of parental NIH/3T3 proliferation compared to that observed in the presence of vehicle.
- An influence on the proliferation rate by the MEK inhibitor, PD 98059 was observed only when cells were incubated in the presence of low concentrations of fetal calf serum (0.1 - 1 %).
- a method of inhibiting or reducing cell proliferation in a human or mammal in need of such treatment comprises the steps of administering to a patient in need thereof a composition comprising a Rsk specific inhibitor, wherein the inhibitor is selected from the group of small-molecules, interference RNA, antisense RNA, antibodies and purified natural products comprising flavonoid compounds represented by the general structure of Formula I.
- a method for inhibiting the growth of neoplastic cells comprises the steps of administering to the human or mammalian patient a Rsk specific inhibitory composition in an amount effective to decrease or inhibit Rsk activity in the target cells.
- the Rsk specific inhibitory composition comprises a compound represented by the general structure:
- Ri, R 2; and R 3j are independently selected from the group consisting of hydroxy -OCOR t , -COR 4 , C ⁇ -C alkoxy, -O-glucoside and -O-rhamnoside
- R 5 , R6,R 7 , R 8 and R 9 are independently selected from the group consisting of H, hydroxy -OCORt, - COR t , C 1 -C 4 alkoxy, -O-glucoside and -O-rhamnoside, and t is H or C ⁇ -C 4 alkyl.
- the method comprises administering a compound of Formula I, wherein Ri, R 2 and R 3 are independently selected from the group consisting of hydroxy and -OCOR , R 5 and R 9 are each H, Rj, R 7 , and R 8 are independently selected from the group consisting of H, -OR 4 , -OCOR t , and -COR4 and t is H or methyl.
- Ri and R 2 are independently selected from the group consisting of hydroxy, -CORi, C1-C4 alkoxy and -OCOCH 3 , R 3 is -OCOCH 3 , t is H or methyl, R 5 , R 8 and R 9 are each H, R ⁇ is H or hydroxy, and R 7 is hydroxy.
- Ri and R 2 are independently selected from the group consisting of hydroxy and - OCOCH 3 , R 3 is -OCOCH 3 , R 5 , R 8 and R 9 are each H, R 6 is H or hydroxy, and R 7 is hydroxy.
- Ri, R 2 and R 3 are independently selected from the group consisting of hydroxy, -OCOCH 3 , -COCH 3 , C ⁇ -C 4 alkoxy, -O-glucoside and -O- rhamnoside, R 5 , Re, R 8 and R 9 are each H and R 7 is hydroxy.
- Ri and R 2 are independently selected from the group consisting of hydroxy and -OCOCH 3 , R 3 is -OCOCH 3 , R 5 , R 6 , R 8 and R 9 are each H and R 7 is hydroxy.
- the Rsk specific inhibitory composition comprises an extract of Forsteronia refracta or Zingiber zerumbet.
- the Rsk specific inhibitory composition comprises a compound represented by the general structure:
- R is H or OH
- Ri, R and R 3 are independently selected from the group consisting of hydroxy, -OCORi, -CORi, C1-C4 alkoxy, -O-glucoside and -O- rhamnoside, and t is H or C ⁇ -C 4 alkyl.
- the composition comprises £ compound of Formula II wherein R is H, R 3 is -OCOCH 3 and Ri and R 2 are independently selected from the group consisting of hydroxy and -OCOCH 3 .
- a method for inhibiting the growth of neoplastic cells through the use of oligonucleotide agents comprises the steps of administering to a patient a Rsk specific inhibitory composition comprising an anti-sense oligonucleotide or interfering oligonucleotide directed against Rskl, Rsk2, Rsk3 or Rsk4.
- a Rsk specific inhibitory composition comprising an anti-sense oligonucleotide or interfering oligonucleotide directed against Rskl, Rsk2, Rsk3 or Rsk4.
- Interfering oligonucleotides include RNA interference molecules (RNAi)s as well as the DNA sequences encoding for such RNAi.
- RNAi in mammalian systems includes the presence of short interfering RNA (siRNA) or short hai ⁇ in RNA (shRNA).
- siRNA typically consists of 19-22nt double-stranded RNA molecules
- shRNA typically consists of 19-29nt palindromic sequences connected by loop sequences, that mimic the structures of micro RNAi.
- larger or smaller nucleic acid sequences than the ranges cited above can be used for the siRNA and shRNA constructs.
- siRNA construct comprising a dsRNA that further comprises a 3' two nucleotide overhang off the sense and antisence strands as described in Elbashir and Tuschl (2001). Genes & Dev. 15: 188-200.
- siRNA and shRNA can be introduced into target cells using standard nucleic acid constructs and techniques known to those skilled in the art. For example, a stable system for expressing siRNA or shRNA has been previously described and utilized to generate transgenic animals (Hasuwa et al. FEBS Lett 532, 227-30 (2002), Rubinson et al.
- RNAi sequences in cells are commercially available from Ambion, Inc.
- antisense and interference RNAs should allow for the design of antisense or interference RNAs that are specific for the individual Rsk isotypes. Since the various Rsk isotypes demonstrate differences in tissue distribution and embryonic expression there may be therapeutic advantages to inhibiting one Rsk isotype verses another.
- RNAi interfering RNA construct
- the interfering RNA (RNAi) construct comprises a nucleic acid sequence selected from the group consisting of
- AAGAAGCUGGACUUCAGCCGU (SEQ IDNO: 5)
- GGCCACACUGAAAGUUCGA (SEQ ID NO 10) ACGUGAUAUCUUGGUAGAG (SEQ ID NO 11) UAUCUUGGUAGAGGUUAAU (SEQ ID NO 12) GAUUUGUUUACACGCUUAU (SEQ ID NO 13) UUUGUUUACACGCUUAUCC (SEQ ID NO 14) ACUUGCACUUGCUUUAGAC (SEQ ID NO 15) GGUCACAUCAAGUUAACAG (SEQ ID NO 16) AAGAGUCUAUUGACCAUGA (SEQ ID NO 17) AGAGUCUAUUGACCAUGAA (SEQ ID NO 18) GAGUCUAUUGACCAUGAAA (SEQ IDNO 19) GUUAAUCGUCGAGGUCAUA (SEQ ID NO 20) GUGCUGACUGGUGGUCUUU (SEQ ID NO 21) AGCGAAAUCCUGCAAACAG (SEQ IDNO: 22) AUCCUGCAAACAGAUUAGG (SEQ ID NO: 23) UCCUGCAAACAGAU
- UCCAAACAUUAUCACUCUA (SEQ ID NO: 32) ACAUUAUCACUCUAAAGGA (SEQ ID NO: 33) CAUUAUCACUCUAAAGGAU (SEQ IDNO: 34) UUAUCACUCUAAAGGAUGU (SEQ IDNO: 35) UCACUCUAAAGGAUGUAUA (SEQ IDNO: 36)
- UGUGUAUGUAGUAACAGAA SEQ IDNO: 37
- UGUGGAUGAAUCUGGUAAU SEQ ID NO: 38
- UCUGGUAAUCCGGAAUCUA SEQ IDNO: 39
- AAAUGGUCUUCUCAUGACU SEQ ID NO: 40
- CAAUGCUUACCGGUUACAC SEQ IDNO: 41
- RNAi comprises a sequence selected from the group consisting of SEQ ID NO: 5-47 linked to its complementary sequence either by a covalent linkage or simply by hydrogen bonding.
- RNAi comprises a sense and anti-sense RNA sequences that are covalently bound to one another by a linking sequence.
- the linking sequence is non-complementary to either of the adjoining sequences, thus allowing the formation a stem loop structure upon hybridization of a sequence of SEQ ID NO: 5-47 to its complementary sequence.
- the Rsk specific inhibitory composition comprises an antibody that is specific for Rsk, and in one embodiment the antibody is specific for a Rsk isotype selected from the group consisting of Rskl, Rsk2, Rsk3 and Rsk4.
- the Rsk specific antibody is directed against the adenosine interacting loop of a Rsk enzyme, including for example, the AIL ofa Rsk isotype selected from the group consisting of Rskl, Rsk2, Rsk3 and Rsk4.
- Antibodies suitable for use as Rsk specific inhibitory compounds include both monoclonal and polyclonal antibodies as well as recombinant proteins comprising the binding domains, as wells as fragments, including Fab, Fab', F(ab) 2 , and F(ab') 2 fragments.
- the Rsk specific inhibitory compounds/compositions of the present invention can be further combined with pharmaceutically acceptable carriers and other therapeutic compounds (such as anti-tumor agents) to provide therapeutic pharmaceutical compositions for treating a wide range of diseases that are associated with inappropriate Rsk activity.
- a composition comprising a Rsk specific inhibitor and an anti-tumor agent is provided.
- the composition may further include a pharmaceutically acceptable canier.
- the Rsk specific inhibitor is an anti-sense oligonucleotide or an interfering oligonucleotide wherein the anti-sense oligonucleotide and interfering oligonucleotide comprise a nucleic acid sequence that is complementary to a nucleic acid sequence present in a Rsk gene, including Rskl, Rsk2, Rsk3 or Rsk4.
- the presently disclosed Rsk inhibitors are used to treat various neoplastic diseases, including cancers such as prostate and breast cancer.
- the inhibitors of the present invention inhibit Rsk specifically in situ without toxic effects, the inhibitors can also be used as therapeutic interventions in non-terminal diseased states such as epilepsy in which the MAPK signaling pathway is improperly regulated.
- the Rsk- specific inhibitors of the present invention are used to treat cancer and neurological disorders such as epilepsy.
- the Rsk specific inhibitors of the present invention can inhibit the growth rate of MCF-7 cells, which are more representative of human cancers than the Ha-Ras transformed cell line.
- MCF-7 cells which are more representative of human cancers than the Ha-Ras transformed cell line.
- SL0101-1 inhibited proliferation of MCF-7 cells but had no effect on the growth of the normal breast cell line, MCF-10A, even though SLOlOl-l prevented the PDB-induced pl40 phosphorylation in MCF-10A cells (Fig. 8A).
- SLO 101-1 inhibits the growth rate of MCF-7 cells at an efficacy that parallels its ability to suppress Rsk activity in vivo.
- SLO 101 did not inhibit the catalytic activity of Protein Kinase C (PKC), RAF, MEK, or MAPK because these kinases are essential to cause phosphorylation of Rsk in cells stimulated with PDB.
- PKC Protein Kinase C
- RAF RAF
- MEK MEK
- MAPK MAPK
- SL0101 is a Rsk-specific inhibitor in situ as well as in vitro and can be used as an investigative tool for defining the function of Rsk in situ.
- SL0101-1 has also been found to completely inhibit the proliferation of LNCaP cells (an androgen-dependent human prostate line), see Fig. 8B. This result suggests that the Rsk inhibitors of the present invention can be used to treat prostate cancer.
- one aspect of the present involves screening individuals for elevated Rsk protein levels and/or Rsk activity (relative to general population levels) as a means of identifying patients that may benefit from Rsk specific inhibitory therapy (i.e. using Rsk levels as a "therapeutic indicator").
- Rsk specific inhibitory therapy i.e. using Rsk levels as a "therapeutic indicator”
- individuals that have cancer or suffer from a neurological disorder may have elevated Rsk levels or activity, and it is anticipated that such patients would benefit from therapy that includes the administration of a Rsk inhibitor.
- the present Rsk specific inhibitors can be used to treat cancer (such as breast cancer) either by using the inhibitors as the sole therapeutic agent or in combination with other anti-tumor agents.
- Rsk protein levels and/or Rsk activity can be used as a therapeutic indicator, and to monitor the effectiveness of a therapeutic treatment, during or after completion of the treatment, allowing for modification of the dosage or other factors to maximize efficacy of the treatment.
- a diagnostic method for detecting neoplastic cells comprises the steps of measuring a Rsk quantification factor, in a biological sample isolated from an individual, and determining if the Rsk quantification factor is elevated relative to an internal or external standard.
- the Rsk quantification factor is any component that relates to the expression or activity of Rsk. In other words this may include mRNA levels or protein levels as well as enzymatic activity which may or may not conelate with Rsk protein levels.
- an elevated Rsk quantification factor i.e. elevated Rsk nucleic acid quantity, Rsk protein quantity or Rsk activity
- the biological sample used for measuring the Rsk quantification factor will comprise a tissue or cell sample recovered from an individual, for example during a biopsy.
- blood or serum samples can also be screened for the presence of Rsk nucleic acid sequences or peptides.
- Analysis of the biological sample to quantitate the Rsk content of the sample can be conducted using standard techniques known to those skilled in the art.
- Overexpression of Rsk can be detected either by determining cellular nucleic acid concentrations or by determining cellular Rsk protein concentrations. This includes the use of in situ analysis as well as the purification of the Rsk nucleic acid or protein.
- the amount of Rsk nucleic acid present in the sample can be determined using labeled complementary Rsk nucleic acid sequences and standard
- the amount of Rsk protein present in the sample can be determined through the use of antibodies that are specific for Rsk epitopes or through the use of other analytical techniques.
- the Rsk protein is purified from the biological sample, and the Rsk kinase activity of the recovered material is determined through the use of an in vitro kinase assay.
- the kinase activity is measured through the use of phospohospecific and/or nonphospohospecific antibodies directed against a Rsk substrate after conducting a kinase assay.
- the Rsk activity is determined by isolating Rsk protein from the biological samples, conducting in vitro kinase assays and determining the rate of formation of phophorylated substrate.
- the amount of Rsk protein is determined by contacting the Rsk protein with a labeled antibody specific for Rsk protein, removing the non-bound and non-specific antibody; and quantifying the amount of label remaining to determine the amount of Rsk protein present.
- the amount of Rsk nucleic acids present in the biological sample is determined by contacting the nucleic acids of the sample with a labeled Rsk complementary nucleic acid probe, removing the non-bound and non-specific probe; and quantifying the amount of label remaining to determine the amount of Rsk nucleic acid.
- the amount of Rsk detected in the biological sample is measured against an internal or external standard.
- an internal standard is used to determine if the Rsk nucleic acid or protein is being overexpressed
- the levels detected in the recovered biological sample can be compared to the nucleic acid or protein levels ofa non-Rsk gene that is expressed in the same tissue used for measuring the Rsk levels.
- a house keeping gene is selected as the internal reference, including for example, actin, Ran or some other gene whose level typically does not fluctuate in the cells selected for the biological sample (i.e. the biopsy tissue).
- the detected Rsk activity in the tissue can be compared to another enzymatic activity present in the same tissue used for measuring the Rsk activity.
- a biological sample can be taken from both healthy tissue and the target tissue (e.g. tumor tissue) of the individual, and the Rsk levels/activity can be compared between the healthy tissue and target tissue taken from the same individual.
- the Rsk levels/activity measured in the biological sample recovered from the patient to be screened can be compared to an external standard (i.e. a biological sample derived from another source).
- the external standard constitutes an average of Rsk levels/activity measured from one or more healthy individuals and used to establish a baseline of Rsk activity. Standard curves will be ustilized based on the tissue type and amounts of starting material used. Such standard curves will be used to determine if an individual's Rsk levels are higher than the population's average levels.
- a diagnostic kit for detecting the presence of neoplastic cells comprises reagents for detecting and quantitating the amount of Rsk or Rsk activity present in a biological sample.
- the kit comprises a Rsk quantifying agent selected from the group consisting of a Rsk specific antibody, a nucleic acid sequence complementary to a Rsk gene sequence or a Rsk substrate and reagents for conducting in vitro kinase assays.
- the antibodies or nucleic acids provided with the kit are labeled or reagents are provided for labeling the Rsk specific antibodies or nucleic acid sequences.
- the antibodies, nucleic acids and other reagents can be packaged in a variety of containers, e.g., vials, tubes, bottles, and the like.
- Other reagents can be included in separate containers and provided with the kit; e.g., positive control samples, negative control samples, buffers, etc.
- the kit is further provided with an anti-tumor agent.
- the kit would also be provided with instructional materials for using the reagents to quantitate a Rsk quantification factor.
- SLO 101-1 inhibited proliferation of MCF-7 cells but did not cause cell death (Fig. 8A).
- SL0101-1 when used in combination with activation of the stress pathways, e.g. by serum deprivation, significantly reduced cell viability compared to vehicle control (Fig. 8C).
- Rsk inhibitors may be most effective when combined with other anti-tumor agents and therapies.
- Rsk inhibitors may also be effective at inhibiting the growth of cancers in which the MAPK pathway is overactive as indicated by the result that SLO 101-1 inhibited the proliferation of Ha-Ras transformed cells but not the parental cells.
- a method of treating a neoplastic disease involves a first step of determining if the disease is characterized by elevated Rsk activity. If such elevated Rsk activity is detected, then the patient is treated with one or more of the Rsk inhibitors of the present invention.
- a method for treating a warm blooded vertebrate patient, including humans, afflicted by a neoplastic disease comprises the steps of administering to such a patient and effective amount of a Rsk specific inhibitor.
- the Rsk specific inhibitor can be administered to a patient in need thereof by either administering an extract of Forsteronia refracta, an antibody specific for Rsk, an RNAi oligomer specific for Rsk, an antisense oligomer specific for Rsk or by administering a composition comprising a Rsk specific inhibitor compound having the general structure:
- R is H or OH
- Ri, R 2 and R 3 are independently selected from the group consisting of hydroxy, -OCOR t , -CORi, C 1 -C 4 alkoxy, -O-glucoside and -O- rhamnoside and R 4 is H or C ⁇ -C 4 alkyl.
- the compound has the structure of compound III, wherein R is H or OH, Ri and R 2 are independently hydroxy or -OCOCH 3 and R 3 is -OCOCH 3 .
- the compound has the structure of compound II or III, wherein R is H, Ri and R 2 are independently hydroxy or -OCOCH 3 and R 3 is -OCOCH 3 .
- a method for inhibiting Rsk kinase activity in a subject is provided, as a means of treating an illness associated with inappropriate Rsk activity.
- the inappropriate Rsk activity may constitute overexpression of Rsk protein, excessive Rsk kinase activity or it may represent the expression of Rsk activity in tissues that normally do not express Rsk activity.
- the Rsk specific inhibitors are used to treat a patient diagnosed with a disease characterized by Rsk hyperactivity, including for example, treating neoplastic disease.
- the Rsk specific inhibitors of the present invention are anticipated to have activity as antiviral agents as well as having use for treating neurological diseased states such as epilepsy.
- a method for treating a disease characterized by elevated Rsk activity comprises the steps of administering to a human or other mammal in need thereof, a therapeutically-effective amount of a composition comprising an extract/concentrate from the tissues of Forsteronia refracta.
- the extract or concentrate is selected from the group consisting of a food grade solvent extract, an aqueous extract (such as a methanol extract) and a dried preparation of the plant.
- the extract or concentrate from the tissues of Forsteronia refracta is administered using standard routes in an amount which is effective for specifically inhibiting Rsk activity in the cells of said human or mammal.
- a method of inhibiting Rsk activity as a means of treating a disease state comprises the steps of administering an antibody specific for Rsk, an RNAi oligomer specific for Rsk, an antisense oligomer specific for Rsk or by administering a composition comprising a compound of the general structure:
- Ri, R 2) and R 3; are independently selected from the group consisting of hydroxy -OCOR 4 , -COR 4 , Q-Q alkoxy, -O-glucoside and -O-rhamnoside, R 5 , R 6 ,R 7 , R 8 and R 9 are independently selected from the group consisting of H, hydroxy -OCOR 4 , - CORi, C ⁇ -C alkoxy, -O-glucoside and -O-rhamnoside, and * is H or C ⁇ -C 4 alkyl.
- the method comprises the steps of administering a compound of Formula I, wherein R ls R 2 and R 3 are independently selected from the group consisting of hydroxy and -OCOIU, R 5 and R 9 are each H, R ⁇ , R 7 , and R 8 are independently selected from the group consisting of H, -OR 4 , -OCOR 4 , and -CORi and R is H or methyl.
- R is H or OH
- Ri, R 2 and R 3 are independently selected from the group consisting of hydroxy, -OCOR- t , -COR , C ⁇ -C 4 alkoxy, -O-glucoside and -O- rhamnoside and R 4 is H or C 1 -C 4 alkyl.
- the method comprises administering a compound represented by Formula II or III, wherein R is H or OH, Ri and R 2 are independently hydroxy or -OCOCH 3 and R 3 is -OCOCH 3 .
- the compound has the structure of compound II or III, wherein R is H, Ri and R 2 are independently hydroxy or -OCOCH 3 and R 3 is -OCOCH 3 .
- the Rsk inhibitory compositions of the present invention can be administered either orally, parenterally, by inhalation or transdermally .
- Rsk inhibiting composition is administered locally by injection or by an implantable time release device.
- the compounds can be administered as a liquid solution, powder, tablet, capsule or lozenge.
- the compounds can be used in combination with one or more conventional pharmaceutical additives or excipients used in the preparation of tablets, capsules, lozenges and other orally administrable forms.
- the derivatives of the present invention can be admixed with saline solutions and/or conventional IV solutions.
- One embodiment of the present invention is directed to pharmaceutical compositions comprising an Rsk inhibitory extract of Forsteronia refracta and a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier can be selected from among the group consisting of excipients, disintegrating agents, binders and lubricating agents.
- the present invention provides a pharmaceutical composition comprising a flavonoid of the general formula I, II or III as defined above and a pharmaceutically acceptable carrier or diluent. The amount of the pharmaceutical agent suitable for administration will be in accordance with standard clinical practice.
- a method for screening for Rsk inhibitors comprises the steps of contacting a kinase substrate with Rsk in the presence ofa potential inhibitory compound for a predetermined length of time under conditions normally permissive for kinase activity. The reaction is then stopped and the amount of phosphorylated substrate is quantitated.
- a control reaction substrate and enzyme without the potential inhibitory compound
- the potential inhibitory compound is also added to a reaction containing a kinase substrate and a kinase other than Rsk to determine if the inhibitory activity of the compound is specific for Rsk.
- HTS high throughput screening
- ELISAs Enzyme-Linked Immunosorbent Assays
- Rsk represents the class of Ser/Thr kinases
- focal adhesion kinase (FAK) represents the Tyr kinases
- extracellular-signal regulated kinase 2 (ERK2) represents the Pro-directed Ser kinases.
- the ELISAs utilize horseradish peroxidase (HRP)-conjugated phosphospecific antibodies or phosphospecific antibodies in combination with HRP-conjugated secondary antibodies.
- HRP horseradish peroxidase
- Purified, recombinant substrate (approximately lug) was adsorbed to the bottom of each well in a 96 well plate.
- the reaction was initiated by the addition of purified, recombinant kinase (approximately 5 nM) in the appropriate buffer contain in 10 ⁇ M ATP. After 10 to 45 minutes the reaction was terminated by addition of EDTA.
- the wells were incubated with the appropriate antibodies, washed and the amount of chemiluminesence determined. All assays measured the initial velocity of reaction. The data was obtained from fully automated assays using the Tecan GENESIS Workstation 150 with integrated Tecan Ultra Reader.
- the Z' factor of an assay is a statistical characteristic of the quality of the assay with respect to the dynamic range and data variation of the signal measurements.
- a Z' factor equal to 1 represents the ideal assay with no background and no deviation of signal, whereas a Z'. 0.5 indicates that the signal window is small to non-existent.
- the Z' of the HTS ELISAs reported herein is -0.8, substantially higher than other HTS assays developed for the identification of kinase inhibitors. These HTS ELISAs were successfully used to screen a botanical extract library. Measurements of kinase activity with and without the presence of various extracts were compared to identify specific inhibitors of Rsk activity. Each plate in the screen contained 80 extracts as well as controls. These controls ensure that there was no plate-to-plate variation in the screen.
- the column was eluted successively with CH 2 C1 2 , 99: 1 CH 2 Cl 2 -MeOH, 95:5 CH 2 Cl 2 -MeOH, 90:10 CH 2 Cl 2 -MeOH and MeOH to give five fractions.
- the 95:5 CH 2 Cl 2 -MeOH, 90:10 CH 2 Cl 2 -MeOH and MeOH fractions showed the same or stronger activity than the starting material.
- the 95:5 CH 2 Cl 2 -MeOH fraction was fractionated repeatedly on a C ⁇ 8 reverse phase HPLC column (250'10 mm); elution was carried out with 65:35 MeOH-H 2 O and UV detection was at 265 nm.
- SL0101-1 and SL0101-2 Two compounds, SL0101-1 and SL0101-2 were obtained as amo ⁇ hous pale yellow powders. On the basis of its ! H NMR spectrum and positive APCI-MS, SL0101-1 was found to be kaempferol 3- ⁇ -L-(3",4"-diacetyl) rhamnopyranoside and SL0101-2 was proved to be kaempferol 3- ⁇ -L- (2",4"-diacetyl) rhamnopyranoside.
- the 90: 10 CH 2 Cl 2 -MeOH fraction from the above diol column was also fractionated repeatedly on a C ⁇ 8 reverse phase HPLC column using 45:55 H 2 0-MeOH as the eluant and UV detection at 275 nm.
- the active constituent, SLO 101-3 was obtained as an amo ⁇ hous powder.
- the compound On the basis of its 1H NMR and 13 C NMR data, the compound was found to be kaempferol-3- ⁇ -L- (4"-acetyl) rhamnopyranoside (SL0101-3).
- the in vitro IC50 was determined to be less than 100 nM for SLO 101-1 (Fig.
- Purified SLO 101-1 is specific for inhibition of Rsk activity compared to p70 S6K and Mskl and is competitive with respect to ATP.
- the specificity of purified SLO 101-1 for Rsk is indicated by the data presented in Fig. 10. That data represent experiments wherein vehicle or inhibitor (5 ⁇ M) was added to the kinase mix containing 5 nM of the indicated purified kinases. The reaction proceeded for 30 mins at room temperature and the data normalized to the kinase activity obtained in the presence of vehicle. The ATP concentration was 10 ⁇ M. Phosphorylation of the substrate was detected by ELISA.
- Polyamide 6S (pour density 0.25 g/mL, a product of Riedel-de Haen, Germany) was obtained from Crescent Chemical Co. Lichroprep diol (40-63 ⁇ m) is a product from EM Industries, Inc. A Kromasil Q 8 reverse phase column (250 x 10 mm, 5 ⁇ m) for HPLC was obtained from Higgins Analytical Inc. H NMR spectra were measured on General Electric QE 300, GN-300 NMR or Varian unity INOVA-500 spectrometers. Mass spectra were recorded on a Finnigan MAT 4600 mass spectrometer. Wood stem and stem bark of Forsteronia refracta was soaked three times with methanol at room temperature.
- the resulting methanol solutions were combined and concentrated under diminished pressure to afford the crude extract.
- the crude extract (888 mg) was applied to a 40-g polyamide 6S column, which was washed successively with 150 mL each of H 2 0, 1:1 H 2 O- MeOH, 9:1 CH 2 Cl 2 -MeOH, 1:1 CH 2 Cl 2 -MeOH and 9:1 MeOH-NH 4 OH to afford five fractions.
- the 1 : 1 CH 2 Cl 2 -MeOH fraction (126.7 mg) showed stronger inhibition of Rsk than the starting original extract.
- the 1:1 CH 2 C1 2 - MeOH fraction was further fractionated on a 30-g diol gel column.
- the column was washed, respectively, with 150 mL each of CH 2 C1 2 , 99:1 CH 2 Cl 2 -MeOH, 95:5 CH 2 Cl 2 -MeOH, 90:10 CH 2 Cl 2 -MeOH and MeOH to give five fractions.
- the 95:5 CH 2 Cl 2 -MeOH (38.1 mg) fraction showed the same or stronger activity than the starting material.
- the 95:5 CH 2 Cl 2 -MeOH fraction (4 mg) was fractionated repeatedly on a Cis reverse phase HPLC column (250 x 10 mm); with elution was 65:35 MeOH-H 2 O at a flow rate of 3 mL/min, with UN detection at 265 nm.
- SL0101-1 (2 mg) was obtained as an amo ⁇ hous pale yellow powder. From the IH ⁇ MR and positive APCI-MS, SL0101-1 was confirmed to be kaempferol 3- ⁇ -L-(3",4"-diacetyl) rhamnopyranoside.
- Rsk contains two non-related kinase domains in a single polypeptide chain.
- the amino-terminal kinase domain ( ⁇ TKD) is mostly closely related to p70 S6K whereas the carboxyl-terminal kinase domain (CTKD) is most similar to the calmodulin-dependent protein kinases.
- CTKD carboxyl-terminal kinase domain
- Regulation of Rsk is complex and requires a cascade of phosphorylations resulting from the actions of MAPK, the CTKD of Rsk itself, and 3-phosphoinositide-dependent protein kinase-1.
- the ⁇ TKD phosphorylates exogenous substrates whereas the only known function of the CTKD is autophosphorylation.
- the extent of phosphorylation was determined using phosphospecific antibodies directly labeled with horseradish peroxidase (HRP)-conjugated or phosphospecific antibodies in combination with HRP-conjugated secondary antibodies. All assays measured the initial reaction velocity and maximum activity was measured in the presence of vehicle. Assays were performed in the presence of vehicle, 2 ⁇ M SL0101-1 or a known non-specific inhibitor, 2 ⁇ M Ro 318220. SLO 101-1 potently inhibited the isolated Rsk2 ⁇ TKD, indicating that inhibition of Rsk occurs through competition with ATP for the nucleotide-binding site of the ⁇ TKD.
- HRP horseradish peroxidase
- SLOlOl-l was ⁇ 3-fold less effective in inhibiting the Rsk2-AIL mutant in comparison to wild type Rsk2 (Fig. 6B). Therefore, the unique adenosine-interacting loop of Rsk is a major determinant for SLOlOl-l binding. However, the mutation did not completely abolish inhibition by SLO 101 - 1 , indicating the presence of additional points of contact.
- the isozyme specificity of SLO 101-1 was examined.
- the primary structure of the NTKDs of the Rsk isoforms 1-3 are highly related, sharing 87% identity and each contain the unique adenosine-interacting loop.
- HA-tagged proteins were immunoprecipitated from the lysates of EGF-stimulated BHK21 cells transiently transfected with the indicated HA-tagged constructs (Rskl, Rsk2 and Rsk3). Assays were performed as described immediately above. Remarkably, although SLO 101-1 potently reduced Rskl and Rsk2 activity, the Rsk3 activity was only partially inhibited (Fig. 6B). Thus, the adenosine interacting loop is necessary but not sufficient for conferring
- SL0101-1 inhibits Rsk in intact cells
- phosphorylation of pl40, a Rsk substrate of unknown function was examined in a human breast cancer cell line, MCF-7.
- MCF-7 and MCF-10A cells were pre-incubated with vehicle, 50 ⁇ M U0126 or the indicated concentration of SL0101-1 for 3 hr (Fig. 8D).
- Cells were treated with 500 nM PDB for 30 min prior to lysis. Protein concentration of lysates was measured and lysates were electrophoresed, transfened and immunoblotted. Equal loading of lysate is demonstrated by the anti-Ran immunoblot.
- SLOlOl-l Pre-incubation of cells with 100 ⁇ M SLOlOl-l abrogates phorbol dibutyrate (PDB)-inducedpl40 phosphorylation as does 50 ⁇ M U0126, a MEK inhibitor.
- PDB phorbol dibutyrate
- SLOlOl-l does not effect the phosphorylation of Rsk2, as indicated by the reduced electrophoretic mobility of Rsk2, nor the activation of MAPK, as detected by the anti-active MAPK antibody (see Fig. 8D). Therefore, SLOlOl-l does not inhibit upstream kinases necessary for PDB-stimulated Rsk phosphorylation, namely MAPK, MEK, Raf and PKC.
- SLO 101-1 could inhibit the growth rate of MCF-7 cells, which are more representative of human cancers than the Ha-Ras transformed cell line.
- MCF-7 cells which are more representative of human cancers than the Ha-Ras transformed cell line.
- SL0101-1 inhibited proliferation of MCF-7 cells but had no effect on the growth of the normal breast cell line, MCF-10A (Fig. 8A), even though SL0101-1 prevented the PDB-induced pl40 phosphorylation in MCF-10A cells (Fig. 8D).
- SLO 101-1 inhibits the growth rate of MCF-7 cells at an efficacy that parallels its ability to suppress Rsk activity in vivo.
- siRNA short, interfering RNAs
- siRNAs duplex siRNAs to a sequence in the bluescript plasmid (Control) or to Rskl and Rsk2 were transfected into MCF-7 cells.
- the sense strand for Rskl has the sequence AAGAAGCUGGACUUCAGCCGU (SEQ ID NO: 5), whereas the sense strand for Rskl has the sequence AACCUAUGGGAGAGGAGGAGA (SEQ ID NO: 6).
- Medium was replaced 24 hr post-transfection and the cells incubated for an additional 48 hr prior to measuring cell viability.
- a combination of siRNAs to both Rskl and Rsk2 was effective in reducing MCF-7 proliferation (Fig.
- Glutathione-S-transferase (GST)-fusion protein (1 g) containing the sequence - RRRLASTNDKG (SEQ ID NO: 3, for serine/threonine kinase assays) or -VSVSETDDYAEIIDEEDTFT (SEQ ID NO: 4, for tyrosine kinase assays) was adsorbed in the wells of LumiNunc 96-well polystyrene plates (MaxiSo ⁇ surface treatment). The wells were blocked with sterile 3% tryptone in phosphate buffered saline and stored at 4°C for up to 6 months.
- kinase (5 nM) in 70 ⁇ l of kinase buffer (5 mM -glycerophosphate pH 7.4, 25 mM HEPES pH 7.4, 1.5 mM DTT, 30 mM MgCl 2 , 0.15 M NaCl) was dispensed into each well.
- Compound at indicated concentrations or vehicle was added, and reactions were initiated by the addition of 30 ⁇ l of ATP for a final ATP concentration of 10 ⁇ M unless indicated otherwise. Reactions were terminated after 10 to 45 min by addition of 75 ⁇ l of 500 mM EDTA, pH 7.5. All assays measured the initial velocity of reaction.
- Cell Culture For proliferation studies cells were seeded at 2500 to 5000 cells per well in 96 well tissue culture plates in the appropriate medium as described by American Type Culture Collection. After 24 hr, the medium was replaced with medium containing compound or vehicle as indicated. Cell viability was measured at indicated time points using CellTiter-GloTM assay reagent (Promega) according to manufacturer's protocol. For in vivo inhibition studies, cells were seeded at 2.5 X10 5 cells/well in 12 well cell culture clusters. After 24 hr, the cells were serum starved for 24 hr then incubated with compound or vehicle for 3 hr prior to a 30 min PDB stimulation. Cells were lysed as previously described( J. Biol. Chem.
- NIH/3T3 cells were seeded on LABTEK II chamber slides (Nalge) at a density of 1 XI 0 4 cells/well. After 24 hr, fresh medium was added the indicated compounds or vehicle. Images were taken 48 hr after treatment at a magnification of 20X.
- Custom oligonucleotides to Rskl (AAGAAGCUGGACUUCAGCCGU; SEQ ID NO: 5 and Rsk2 (AACCUAUGGGAGAGGAGGAGA; SEQ ID NO: 6) mRNA (Dharmacon Research Inc.) and TransIT-TKO® siRNA Tranfection Reagent (MIR2150, Minis Co ⁇ oration) were used for the gene silencing studies.
- MCF-7 cells were seeded at a density of 1.25X10 4 cells per well in 24 well cell culture clusters. After 24 hr, fresh medium was added containing 25 nM oligonucleotide and transfection reagent according to manufacturer's protocol. The transfection medium was replaced after 24 hr. Cells were incubated for an additional 48 hr prior to cell viability measurement.
- Prostate cancer is the second most common cancer in men and approximately one in six men will be diagnosed with the disease.
- Early stage prostate cancer is frequently dependent on the hormone, androgen. Androgen action is mediated through interaction with the androgen receptor, a member of the superfamily of ligand-activated transcription factors. Inhibition of androgen receptor activity by pharmacological or surgical interventions that reduce androgen concentration can result in prostate tumor regression.
- the tumors become androgen-independent, which often leads to a fatal outcome.
- Treatment options are confined to conventional chemotherapy because of the lack of specific drug targets associated with androgen-independent prostate cancer. Thus, elucidation of the mechanisms that result in the transition of prostate cancer from an androgen-dependent to androgen-independent state will greatly facilitate the identification of more effective therapies.
- mitogen-activated protein kinase AMAP-activated protein kinase (MAPK) activity has been conelated to prostate cancer progression in human tumors. This enhanced activity is most likely due to the increase in growth factors and receptors that are known to occur. Activation of growth factor receptors enhance MAPK activity via a kinase cascade that is regulated by the small GTP-binding protein, p21Ras.
- the biological actions of the Rsks are not well characterized partly because until recently there were no known inhibitors of Rsk that did not also inhibit MAPK activity.
- the first Rsk-specific inhibitor, SL0101-1 has now been isolated. As described in Example 4, SL0101-1 inhibits the proliferation of the breast cancer cell line, MCF-7, without preventing the proliferation of a normal breast cell line, MCF-10A. Furthermore, in NIH 3T3 fibroblasts, SL0101 reduces the growth of a Ha-Ras- transformed line but not of the untransformed parental cells. It is believed that SL0101 specifically inhibits the growth of transformed cells because transformed cells preferentially depend on the Rsk pathway to regulate proliferation. These results provide the first demonstration that the Rsk family through the regulation of its downstream effectors is involved in the control of cancer cell proliferation. Relatively few downstream effectors of Rsk have been identified.
- Rsk is known to phosphorylate and regulate the activity of a number of transcription factors, the pro-apoptotic protein, BAD, and the mitotic checkpoint kinase, BUB1. Determining which Rsk substrates play a key role in cancer cell proliferation will undoubtedly lead to the discovery of novel drug targets for cancer therapy.
- Rsk2 Overexpression of the isoform 2 of the Rsk family (Rsk2) also enhances the transcriptional activity of the estrogen receptor (ER ⁇ ) and the androgen receptor (AR).
- ER ⁇ estrogen receptor
- AR androgen receptor
- Rsk2 enhanced both ligand-dependent and ligand-independent ER-mediated transcription in MCF-7 cells, a human breast cancer cell line (Fig. 9A). Additionally, Rsk2 enhances the ligand-dependent and lingand-independent transcription of AR-mediated transcription in LNCaP cells, a human prostate cancer cell line (Fig. 9B).
- TBDPS tert- butyldiphenylsilyl
- THF tetrahydrofuran
- EDCI l-(3-dimethylaminopropyl)-3- ethylcarbodiimide
- DMAP 4-dimethylaminopyridine
- TSOH 4-toluene sulfonic acid
- DMF dimethylformamide
- Bn benzyl
- MTBE methyl tert-butyl ether.
- R 0 H or OTBDPS
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP03760343A EP1539781A4 (en) | 2002-06-12 | 2003-06-12 | Rsk inhibitors and therapeutic uses thereof |
US10/517,328 US20050233985A1 (en) | 2003-06-12 | 2003-06-12 | Rsk inhibitors and therapeutic uses thereof |
AU2003251513A AU2003251513A1 (en) | 2002-06-12 | 2003-06-12 | Rsk inhibitors and therapeutic uses thereof |
CA002488864A CA2488864A1 (en) | 2002-06-12 | 2003-06-12 | Rsk inhibitors and therapeutic uses thereof |
US11/524,159 US20070049539A1 (en) | 2002-06-12 | 2006-09-20 | Rsk inhibitors and therapeutic uses thereof |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US38800602P | 2002-06-12 | 2002-06-12 | |
US60/388,006 | 2002-06-12 | ||
US44955303P | 2003-02-24 | 2003-02-24 | |
US60/449,553 | 2003-02-24 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/524,159 Division US20070049539A1 (en) | 2002-06-12 | 2006-09-20 | Rsk inhibitors and therapeutic uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2003105766A2 true WO2003105766A2 (en) | 2003-12-24 |
WO2003105766A3 WO2003105766A3 (en) | 2004-03-11 |
Family
ID=29739979
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2003/018734 WO2003105766A2 (en) | 2002-06-12 | 2003-06-12 | Rsk inhibitors and therapeutic uses thereof |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1539781A4 (en) |
AU (1) | AU2003251513A1 (en) |
CA (1) | CA2488864A1 (en) |
WO (1) | WO2003105766A2 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100590818B1 (en) | 2004-10-11 | 2006-06-19 | 재단법인서울대학교산학협력재단 | The use of direct activation by organic agents of p90 ribosomal S6 kinase 1 RSK1 for prevention and treatment of diabetes, obesity and metabolic syndrome |
WO2006086103A2 (en) * | 2005-01-10 | 2006-08-17 | University Of Virginia Patent Foundation | Synthesis of inhibitors of p90rsk |
WO2006132401A1 (en) * | 2005-06-07 | 2006-12-14 | Banyu Pharmaceutical Co., Ltd. | Method for evaluation of compound using rsk1 |
WO2006134352A1 (en) * | 2005-06-15 | 2006-12-21 | Biosynth As | Method for the synthesis of anthocyanins |
WO2009035987A1 (en) * | 2007-09-13 | 2009-03-19 | Gateway Health Alliances, Inc. | METHODS AND RELATED COMPOSITIONS USING SPECIFIC FLAVONOIDS AND INDANES TO REDUCE WEIGHT AND INHIBIT LIPASE. α-AMYLASE AND α-GLUCOSIDASE ACTIVITY IN MAMMALS |
WO2009040512A2 (en) * | 2007-09-27 | 2009-04-02 | University Court Of The University Of Dundee | Modulation of rsk |
US8318213B2 (en) | 1999-08-27 | 2012-11-27 | Gateway Health Alliances, Inc. | Plant extract mixtures and their uses |
WO2013181742A1 (en) * | 2012-06-04 | 2013-12-12 | Phoenix Molecular Diagnostics Ltd. | Methods of inhibiting rsk for treatment of breast cancer |
US9040673B2 (en) | 2009-08-14 | 2015-05-26 | University Of Virginia Patent Foundation | Synthesis and identification of novel RSK-specific inhibitors |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000066721A2 (en) * | 1999-04-30 | 2000-11-09 | Beth Israel Deaconess Medical Center | Decreasing adipose mass by altering rsk2 activity |
-
2003
- 2003-06-12 EP EP03760343A patent/EP1539781A4/en not_active Withdrawn
- 2003-06-12 CA CA002488864A patent/CA2488864A1/en not_active Abandoned
- 2003-06-12 WO PCT/US2003/018734 patent/WO2003105766A2/en not_active Application Discontinuation
- 2003-06-12 AU AU2003251513A patent/AU2003251513A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000066721A2 (en) * | 1999-04-30 | 2000-11-09 | Beth Israel Deaconess Medical Center | Decreasing adipose mass by altering rsk2 activity |
Cited By (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8318213B2 (en) | 1999-08-27 | 2012-11-27 | Gateway Health Alliances, Inc. | Plant extract mixtures and their uses |
KR100590818B1 (en) | 2004-10-11 | 2006-06-19 | 재단법인서울대학교산학협력재단 | The use of direct activation by organic agents of p90 ribosomal S6 kinase 1 RSK1 for prevention and treatment of diabetes, obesity and metabolic syndrome |
WO2006086103A2 (en) * | 2005-01-10 | 2006-08-17 | University Of Virginia Patent Foundation | Synthesis of inhibitors of p90rsk |
WO2006086103A3 (en) * | 2005-01-10 | 2006-09-28 | Univ Virginia | Synthesis of inhibitors of p90rsk |
US7605241B2 (en) | 2005-01-10 | 2009-10-20 | University Of Virginia Patent Foundation | Synthesis of inhibitors of p90Rsk |
EP2305830A3 (en) * | 2005-06-07 | 2011-08-10 | Msd K.K. | Method for evaluation of compound using RSK1 |
WO2006132401A1 (en) * | 2005-06-07 | 2006-12-14 | Banyu Pharmaceutical Co., Ltd. | Method for evaluation of compound using rsk1 |
EP1892301A1 (en) * | 2005-06-07 | 2008-02-27 | Banyu Pharmaceutical Co., Ltd. | Method for evaluation of compound using rsk1 |
EP1892301A4 (en) * | 2005-06-07 | 2008-09-17 | Banyu Pharma Co Ltd | Method for evaluation of compound using rsk1 |
WO2006134352A1 (en) * | 2005-06-15 | 2006-12-21 | Biosynth As | Method for the synthesis of anthocyanins |
NO338953B1 (en) * | 2005-06-15 | 2016-11-07 | Biosynth As | Process for the synthesis of anthocyanins |
US8513395B2 (en) | 2005-06-15 | 2013-08-20 | Biosynth As | Method for the synthesis of anthocyanins |
US8362090B2 (en) | 2007-09-13 | 2013-01-29 | Gateway Health Alliances, Inc. | Methods and related compositions using specific flavonoids and indanes to reduce weight and inhibit lipase, α-amylase and α-glucosidase activity in mammals |
GB2466400A (en) * | 2007-09-13 | 2010-06-23 | Gateway Health Alliance Inc | Methods and related compositions using specific flavonoids andindanes to reduce weight and inhibit lipase a-amylase and a-glucosidase activity in mammals |
US7659313B2 (en) | 2007-09-13 | 2010-02-09 | Gateway Health Alliances, Inc. | Methods and related compositions using specific indanes to reduce weight and inhibit lipase, α-amylase and α-glucosidase activity in mammals |
US8394860B2 (en) | 2007-09-13 | 2013-03-12 | Gateway Health Alliances, Inc. | Methods and related compositions using specific flavonoids and indanes to reduce weight and inhibit lipase, α-amylase and α-glucosidase activity in mammals |
WO2009035987A1 (en) * | 2007-09-13 | 2009-03-19 | Gateway Health Alliances, Inc. | METHODS AND RELATED COMPOSITIONS USING SPECIFIC FLAVONOIDS AND INDANES TO REDUCE WEIGHT AND INHIBIT LIPASE. α-AMYLASE AND α-GLUCOSIDASE ACTIVITY IN MAMMALS |
WO2009040512A3 (en) * | 2007-09-27 | 2009-12-03 | University Court Of The University Of Dundee | Modulation of rsk |
WO2009040512A2 (en) * | 2007-09-27 | 2009-04-02 | University Court Of The University Of Dundee | Modulation of rsk |
US9040673B2 (en) | 2009-08-14 | 2015-05-26 | University Of Virginia Patent Foundation | Synthesis and identification of novel RSK-specific inhibitors |
WO2013181742A1 (en) * | 2012-06-04 | 2013-12-12 | Phoenix Molecular Diagnostics Ltd. | Methods of inhibiting rsk for treatment of breast cancer |
Also Published As
Publication number | Publication date |
---|---|
EP1539781A2 (en) | 2005-06-15 |
WO2003105766A3 (en) | 2004-03-11 |
AU2003251513A1 (en) | 2003-12-31 |
EP1539781A4 (en) | 2009-12-02 |
CA2488864A1 (en) | 2003-12-24 |
AU2003251513A8 (en) | 2003-12-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20070049539A1 (en) | Rsk inhibitors and therapeutic uses thereof | |
Daemen et al. | Pan-cancer metabolic signature predicts co-dependency on glutaminase and de novo glutathione synthesis linked to a high-mesenchymal cell state | |
Wu et al. | Autotaxin expression and its connection with the TNF-alpha-NF-κB axis in human hepatocellular carcinoma | |
Pogorelcnik et al. | Recent developments of DNA poisons-human DNA topoisomerase IIα inhibitors-as anticancer agents | |
Tseng et al. | Synthesis and anti-inflammatory evaluations of β-lapachone derivatives | |
Xue et al. | Novel myricetin derivatives: Design, synthesis and anticancer activity | |
Yang et al. | Phosphorylations of DEAD box p68 RNA helicase are associated with cancer development and cell proliferation | |
JP4851451B2 (en) | Breast cancer-related gene ZNFN3A1 | |
Yuan et al. | TIPE3 is a regulator of cell apoptosis in glioblastoma | |
US9493813B2 (en) | Modulation of phosphatidylinositol-5-phosphate-4-kinase activity | |
Xu et al. | RNASET2 impairs the sperm motility via PKA/PI3K/calcium signal pathways | |
EP1539781A2 (en) | Rsk inhibitors and therapeutic uses thereof | |
Gu et al. | Peperomin E induces promoter hypomethylation of metastatic-suppressor genes and attenuates metastasis in poorly differentiated gastric cancer | |
Uchiyama et al. | Aristeromycin and DZNeP cause growth inhibition of prostate cancer via induction of mir-26a | |
Dilshara et al. | Methanol extract of Codium fragile inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 and invasiveness of MDA-MB-231 cells by suppressing nuclear factor-κB activation | |
CN112011614B (en) | Application of KMT5A in regulation of glioma stem cell characteristics and glioma diagnosis and treatment | |
Smith et al. | Influence of rhamnose substituents on the potency of SL0101, an inhibitor of the Ser/Thr kinase, RSK | |
Jiang et al. | Multifunctions of CRIF1 in cancers and mitochondrial dysfunction | |
EP3808350A1 (en) | Composition comprising flt3 inhibitor as effective ingredient for inhibiting drug resistance in chronic myelogenous leukemia | |
US11510911B2 (en) | Method for prediction of susceptibility to sorafenib treatment by using SULF2 gene, and composition for treatment of cancer comprising SULF2 inhibitor | |
WO2016008051A1 (en) | Contactin-1 (cntn1) for use in methods of diagnosis and treatment of prostate cancer | |
US20230330111A1 (en) | Active agent combination for treatment of cancer | |
KR101793175B1 (en) | Method for Prediction of Susceptibility to Sorafenib Using SULF2 Gene and Composition for Treating Cancer Containing SULF2 Inhibitor | |
Cao et al. | Erlotinib is effective against FLT3‐ITD mutant AML and helps to overcome intratumoral heterogeneity via targeting FLT3 and Lyn | |
Cataldi et al. | Phosphatidylinositol-3-kinase activation and atypical protein kinase C ζ phosphorylation characterize the DMSO signalling in erythroleukemia cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 10517328 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2488864 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2003760343 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2003760343 Country of ref document: EP |
|
NENP | Non-entry into the national phase in: |
Ref country code: JP |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |