WO2015123617A1 - Procédé pour réduire l'expression du gène bcl2 - Google Patents

Procédé pour réduire l'expression du gène bcl2 Download PDF

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WO2015123617A1
WO2015123617A1 PCT/US2015/015981 US2015015981W WO2015123617A1 WO 2015123617 A1 WO2015123617 A1 WO 2015123617A1 US 2015015981 W US2015015981 W US 2015015981W WO 2015123617 A1 WO2015123617 A1 WO 2015123617A1
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bcl2
motif
imc
hnrnp
compound
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PCT/US2015/015981
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Laurence Hurley
Samantha KENDRICK
Vijay Gokhale
Danzhou Yang
Prashansa AGRAWAL
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Arizona Board Of Regents For The University Of Arizona
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4453—Non condensed piperidines, e.g. piperocaine only substituted in position 1, e.g. propipocaine, diperodon
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a method for treating a clinical condition associated with overexpression of BCL2 gene.
  • the present invention relates to treating a clinical condition associated with overexpression of BCL2 gene by administering a compound that reduces i-motif structure of BCL2 gene.
  • DNA secondary structures such as G-quadruplexes
  • G-quadruplexes are believed to play important roles in cellular processes including transcription.
  • Bioinformatics studies have revealed that G-quadruplex DNA secondary structure-forming sequences are concentrated near transcriptional start sites in promoter, 5 '-UTR, in addition to telomeric regions. These structures have the potential to form during nuclear processes, such as transcription, when local unwinding of duplex DNA produces negative supercoiling and associated torsional stress.
  • G-quadruplexes are proposed to act as molecular switches that turn transcription on or off.
  • G- quadruplexes within the c-MYC, c-KIT, KRAS, VEGF, PDGFA, PDGFR- ⁇ , HIF-la, c-MYB, and h TERT promoter regions have served as molecular targets for altering gene expression.
  • DNA secondary structures to serve as molecular switches likely involves interaction with transcriptional proteins, for example, NM23-H2/nucleoside diphosphate kinase B and nucleolin recognize and interact with the c- MYC G-quadruplex to alter transcription. Transcriptional repression studies have so far focused on the G-quadruplex as a target.
  • B-cell lymphoma gene-2 (B-cell lymphoma gene-2, a pro-survival oncoprotein) has been linked to the development of cancer chemoresistance, particularly for those of lymphocytic origin. It is believed that BCL2 overexpression prolongs cell survival and resistance to apoptosis, despite
  • chemotherapeutic treatment Targeting of BCL2 to increase chemotherapeutic efficacy has been explored with approaches ranging from disruption of BCL2 protein-protein interactions with small molecules, such as ABT-236 and ABT-737, to siRNA-mediated knockdown of mRNA transcript levels.
  • Some aspects of the invention are based on the discovery by the present inventors that BCL2 transcription can be modulated through interaction of small molecules with a putative czs-regulatory element within the BCL2 promoter region, in particular a partially unfolded i-motif called a flexible hairpin.
  • the present inventors have also discovered that the i-motif exists in equilibrium with an unfolded species and that the relative populations of the two species determine the extent of transcriptional activation. Identification of compounds that bind preferentially either to the i-motif or its unfolded form allows for the external manipulation of the two species populations to modulate gene expression. Some embodiments of the invention provide modulation of BCL2 gene transcription by small molecules that target two alternatively folded forms of BCL2 gene in dynamic equilibrium. Yet other embodiments provide a strategy for therapeutic intervention based on shifting the equilibrium populations of BCL2 gene conformational isomers. In one particular embodiment, the method increases formation of the flexible hairpin conformation of BCL2 gene compared to the i-motif conformation of BCL2 gene.
  • the amount of relative increase in flexible hairpin conformation (relative to the amount of flexible hairpin conformation in the absence of a compound that is used to treat a clinical condition) is at least 5%, typically at least 10%, often at least 25%, and more often at least 50%.
  • Figure 1 is a diagram of the BCL2 gene promoter region with the GC-rich element located directly upstream of the PI promoter.
  • Three and one-half sets of two intercalated hemiprotonated cytosine-cytosine base pairs form the i-motif structure, and three-stacked G-tetrads form the G-quadruplex.
  • the group of bases I-VI are involved in base pairing within each of the structures.
  • Group I nucleotides -1486 to -1483, II: -1479 to -1477, III: -1475 to -1471, IV: -1465 to 11463, V: -1459 to -1457 and VI: -1455 to -1452.
  • the yellow, green, red, and blue circles represent the nucleobases cytosine, adenine, guanine, and thymine, respectively.
  • Figures 2A-2D shows NMR results used to identify the two equilibrating species from the BCL2 C-rich strand and effect of IMC-48 and IMC-76 individually and in combination on the population dynamics.
  • the top panel shows the GC base-pairing (blue lines) of the flexible hairpin (left) and the folding pattern of the i-motif (right).
  • the C-runs are numbered I- VI.
  • Figure A is the imino proton region of the ID 1H-NMR spectra of free BCL2 i-motif DNA and its titration with different equivalents of IMC-76.
  • Figure 2B is the imino proton region of the ID 1H NMR spectra of free BCL2 i-motif DNA and its titration with different equivalents (0.5, 1, 2, 3, and 4) of IMC-48 at pH 6.6, 25 °C (left) and 3 °C (right).
  • Figure 2C is the imino proton region of the ID 1H NMR spectra of free BCL2 i-motif DNA and its complexes with two equivalents of IMC-48 and different equivalents of IMC-76 at pH 6.6, 25 °C (left) and 3 °C (right).
  • Spectra 1 are for free DNA; spectra 2-5 are for 1 :2 DNA: IMC-48 complexes titrated with increasing amount of IMC-76, at 0, 2, 4, and 6 equivalents, respectively.
  • Figure 2D is the imino proton region of the ID 1H NMR spectra of free BCL2 i-motif DNA and its complexes with two equivalents of IMC-76 and different equivalents of IMC-48 at pH 6.6, 25 °C (left) and 3 °C (right).
  • Spectra 1 are for free DNA; spectra 2-5 are for 1 :2 DNA:IMC-76 complexes titrated with increasing amount of IMC-76, at 0, 2, 4, and 6 equivalents, respectively.
  • Figures 3A-3E are experimental results of BCL2 downregulation and chemosensitization in lymphoma cell lines.
  • Figure 3 A shows results of Basal BCL2 mRNA levels (left) (*P ⁇ 0.0001) and protein expression (right).
  • Figures 3B and 3C are results of BCL2 mRNA levels in B95.8 (upper), GRANTA-519 (middle), and BJAB (lower) cells in the presence of IMC-76 or IMC-48 (*P ⁇ 0.05), respectively.
  • Figure 3D shows percent caspase-3 activity of B95.8 (upper), GRANTA-519, (middle), and BJAB (lower) cells with IMC-76, etoposide, and ABT-737 relative to untreated (set to 100%) (*P ⁇ 0.04).
  • Figure 3E shows percent caspase-3 activity of GRANTA-519 cells with IMC-76 and IMC-48 with and without cyclophosphamide (Cytoxan) (*P ⁇ 0.04). Significance determined by two-tailed Student's t- test.
  • Figure 4 BCL2 downregulation and chemosensitization in lymphoma in vivo.
  • A BCL2 mRNA levels (left) and BCL2 protein expression (right) of excised GRANTA-519 lymphoid tumors from mice.
  • B Tumor burden of GRANT A-519 xenograft mice (left) and mean mouse weight (right).
  • Figure 5 shows effect of IMC-76 and IMC-48 on cell toxicity in lymphoma and breast cancer cell lines. Percent survival determined by the MTS cytotoxicity assay in response to treatment with IMC-76 (Panel A) for BJAB, B95.8 and GRANTA-519 lymphoma and MCF-7, MCF-7/TAMR, and MDA-MB-231 breast cancer cell lines or IMC- 48 (Panel B) for BJAB and B95.8 lymphoma cell lines at 96 h. Percent survival was calculated relative to untreated controls from three independent experiments.
  • FIG. 6 shows effect of IMC-76 and IMC-48 on BCL-2 protein levels in lymphoma cell lines.
  • lanes 1 and 2 represent untreated and DMSO vehicle cell lysates, respectively.
  • lanes 3 and 4 contain lysates from cells treated with 0.25 and 0.5 ⁇ IMC-76, respectively.
  • panel B lanes 3 and 4 contain lysates from cells treated with 1 and 2 ⁇ IMC-48, respectively.
  • the western blots are representative of three independent experiments with ⁇ -actin as a loading control.
  • Figure 7 shows effect of IMC-76 on BCL2 mRNA levels in various breast carcinoma cell lines.
  • MCF-7 and MDA-MB-231 BCL-2 mRNA levels were normalized to ⁇ -actin and MCF-7/TAMR levels were normalized to GAPDH. *P ⁇ 0.02.
  • Figure 8 Diagram of the BCL2 gene promoter region with the GC-rich element located directly upstream of the PI promoter and targeting with IMC-48 and IMC- 76. The C-rich i-motif-forming sequence is shown. Three and one -half sets of two
  • intercalated hemiprotonated cytosine-cytosine base pairs form the i-motif structure.
  • the lower portion of the figure shows the proposed partial hairpin which is in equilibrium with the i-motif and the proposed binding of IMC-48 and IMC-76 to the i-motif and partial hairpin respectively along with the proposed transcriptional consequences of targeting with IMC-48 and IMC-76.
  • Figures 9A-9D show results confirming hnRNP LL as a BCL2 i-motif-binding protein.
  • Figure 9A shows the effects of siRNA knockdown of hnRNP LL on the BCL2 mRNA level in MCF-7 cells. 50 nM of siRNA to hnRNP LL was added to MCF-7 cells for 72 h and 48 h, respectively. GAPDH was used as an internal control ( **P ⁇ 0.01).
  • Figure 9B shows effect on concentration-dependent binding of hnRNP LL on the BCL2 i- motif-forming oligomer (Py39WT) by EMSA at pH 6.8.
  • Figure 9C is a result of competition EMS A showing BCL2 i-motif-specific binding of hnRNP LL at pH 6.8.
  • Non-labeled (cold) oligomers were incubated with hnRNP LL on ice for 20 min and end-labeled Py39WT was added for 5 min.
  • Figure 9D shows comparative Ka values for hnRNP LL binding to the biotin-Py39WT and biotin-Py39MutT at two different pH levels determined by SPR analysis.
  • Figure 10 is a result of luciferase promoter assay showing that knockdown with hnRNP LL siDNA was dependent on the wild-type sequence in the lateral loops of the i- motif.
  • Three pGL3 constructs of wild-type, Mut5 ' ,3 ' L, and MutCL were co-transfected with pRL-TK for normalization and siRNA to hnRNP LL for 72 h.
  • Final relative luciferase activities were obtained by normalization of the ratio of firefly to renilla to siRNA-untreated control of each construct (****p ⁇ 0.0001, ***P ⁇ 0.001, ns: not significant).
  • Figure 11 shows results of bromine footprinting of the BCL2 i-motif and hnRNP LL complex showing the conformational change of Py39WT induced by hnRNP LL.
  • Py39WT and hnRNP LL were incubated for 5 min at room temperature and bromine generated in situ was added for 30 min.
  • Black and red plots are 0 and 10 ⁇ g of hnRNP LL, respectively.
  • the peaks with the black dots correspond to those where maximum inhibition occurs and include C-runs II and IV and the central loop.
  • the right panel shows the folding pattern of the BCL2 i-motif with that region protected from Br2 cleavage shown in the blue shading.
  • Figure 12 shows the consequences of sequestration of the flexible hairpin or the BCL2 i-motif by IMC-76 (Panel A) and IMC-48 (Panel B) respectively, on the binding of hnRNP LL to the i-motif.
  • Panel A shows EMSA analysis of the competition between IMC- 76 and hnRNP LL for the i-motif (left) and densitometric analysis (right).
  • Panel B shows EMSA analysis of the cooperativity between IMC-48 and hnRNP LL for the i-motif (left) and densitometric analysis (right).
  • IMC-76 or IMC-48 were incubated with Py39WT for 3 h and hnRNP LL was added for 10 min at pH 6.5 before running the 6% native PAGE. Relative band intensities are plotted against IMC-76 or IMC-48 concentrations (right). Species 1 and 2 are proposed to be the i-motif and flexible hairpin, respectively.
  • Figure 13 shows conformational transitions and biological consequences that occur following mutually exclusive binding of IMC-76, IMC-48 and hnRNP LL to the different equilibrating forms of the c-rich strand in the BCL2 promoter.
  • Box A shows the two different major conformational states of the C-rich strand in the BCL2 promoter under different H conditions. Acidic conditions drive formation of the i-motif, and at pH 6.6 there is a conformational mixture of the flexible hairpin and i-motif.
  • the flexible hairpin form is sequestered (A to B), resulting in depletion of the populations of the i-motif species.
  • IMC-48 binds to the central loop of the Bcl-2 i-motif to sequester this species and then the RRMs 1 and 2 of the hnR P LL, which are closely spaced apart, are initially proposed to recognize and bind to both of the lateral loops II and V, which are constrained in a single-stranded form (A to C).
  • RRMs 1 and 2 of the hnR P LL which are closely spaced apart
  • hnRNP LL bound to the alternative conformation of the C-rich strand causes transcriptional activation of BCL2 (D to E).
  • the consequence of competition between IMC-76 and hnRNP LL for the different conformational states of the C-rich strand depletes the population undergoing the transition A to C to D to E and repression of BCL2 gene expression.
  • binding of IMC-48 to the BCL2-i-motif leads to an increased amount of i-motif that is bound by hnRNP LL and transcriptional activation (A to C to D).
  • Figure 14 is SPR sensorgrams for binding of increasing concentrations of hnRNP LL to Py39WT (left) and Py39MutT (right). The pH of the sensorgram is 6.8.
  • Some aspects of the invention provide methods for treating a clinical condition associated with overexpression of BCL2 gene. Such methods include administering to a subject in need of such a treatment a compound that reduces i-motif structure of BCL2 gene, thereby reducing the expression of BCL2 gene. Methods of the invention reduces BCL2 gene expression by at least about 10%, typically at least about 20%, often at least about 30%, more often at least about 40% and most often by at least 50% compared with the level of BCL2 gene expression in the absence of such a compound.
  • the term "about” refers to ⁇ 20%, typically ⁇ 10%>, and often ⁇ 5% of the numeric value.
  • Treating" or “treatment” of a clinical condition includes: (1) preventing the clinical condition, i.e., causing the clinical symptoms of the clinical condition (e.g., disease) not to develop in a mammal that may be exposed to or predisposed to the clinical condition but does not yet experience or display symptoms of the clinical condition; (2) inhibiting the clinical condition, i.e., arresting or reducing the development of the clinical condition or its clinical symptoms; or (3) relieving the clinical condition, i.e., causing regression of the clinical condition or its clinical symptoms.
  • said i-motif structure occurs in the promoter region of BCL2 gene.
  • said compound increases the imino proton area of proton nuclear magnetic resonance (NMR) associated with a flexible hairpin species at pH 6.6 at temperature of about 3 °C to about at least 50%, typically at least 75%, often at least 80% and most often at least 90% when the ratio of said compound to BCL2 DNA is about 4: 1.
  • said compound decrease the imino proton area of proton nuclear magnetic resonance associated with said i- motif structure at pH 6.6 at temperature of about 3 °C to about at least 50%, typically at least 75%, often at least 80% and most often at least 90% when the ratio of said compound to BCL2 DNA is about 4: 1.
  • said compound has a steroid core structure.
  • said compound is of the formula:
  • R 1 is -OR a
  • R 3 is hydrogen or -OR a
  • R 5 is - OR a or -NHR d
  • each R b is independently H or alkyl
  • each of R c and R h is independently alkylene
  • each of R e and R f is independently alkyl or optionally substituted aryl
  • R b is alkyl. In one particular embodiment, R b is methyl.
  • R 5 is -OR a . In one particular embodiment, R a is H. In other embodiments, R 5 is -NHR d .
  • X 2 is -NR b R b , where each R b is indpendetly H or alkyl, such as methyl, ethyl, propryl, isopropyl, t-butyl, sec-butyl, etc.
  • X 2 is optionally substituted heterocyclyl or optionally substitute pyridyl.
  • X 2 is selected from the group consisting
  • R g is methyl. Still in other embodiments, R h is ethylene. In other embodiments, R e and R f together with the nitrogen atom to which they are attached to form an optionally substituted five- or six-membered heterocyclyl. In some particular embodiments R e and R f together with the nitrogen atom to which they are attached t form:
  • R 7 is optionally substituted (heterocyclyl)alkyl; R s is optionally substituted cyclyl or aryl, or a steroidal moiety; and X 3 is -O- or -NH-.
  • R 7 is
  • heterocyclyl of R 7 is piperidinyl or N-methylpiperidinyl.
  • heterocyclyl of R 7 is piperidin-l-yl or N-methylpiperidin-l-yl.
  • X 3 is NH.
  • R 8 is C17-substituted steroid moiety or cyclohexyl, phenyl, or adamantyl, each of which is optionally substituted.
  • R 8 is 17-(6-methylhept- 2-yl)steroid moiety, cyclohexyl, phenyl, adamant- 1-yl, or 2,4,6-trimethylphenyl.
  • Alkyl refers to a saturated linear monovalent hydrocarbon moiety of one to twelve, typically one to six, carbon atoms or a saturated branched monovalent hydrocarbon moiety of three to twelve, typically three to six, carbon atoms.
  • Exemplary alkyl group include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl, tert-butyl, pentyl, and the like.
  • Alkylene refers to a saturated linear divalent hydrocarbon moiety of one to twelve, typically one to six, carbon atoms or a branched saturated divalent hydrocarbon moiety of three to twelve, typically three to six, carbon atoms.
  • alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, and the like.
  • Aryl refers to a monovalent mono-, bi- or tricyclic aromatic hydrocarbon moiety of 6 to 15 ring atoms which is optionally substituted with one or more, typically one, two, or three substituents within the ring structure. When two or more substituents are present in an aryl group, each substituent is independently selected.
  • Exemplary aryl includes, but is not limited to, phenyl, 1-naphthyl, and 2-naphthyl, and the like, each of which can optionally be substituted.
  • Alkyl refers to a moiety of the formula -R b R c where R b is an alkylene group and R c is an aryl group as defined herein.
  • exemplary aralkyl groups include, but are not limited to, benzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, and the like.
  • cycloalkyl and cyclyl are used interchangeably herein and refer to a non-aromatic, typically saturated or mono-unsaturated, monovalent mono-, bi- or tricyclic hydrocarbon moiety of three to fifteen ring carbons.
  • the cycloalkyl can be optionally substituted with one or more, typically one, two, or three, substituents within the ring structure. When two or more substituents are present in a cycloalkyl group, each substituent is independently selected.
  • Exemplary cycloalkyl includes, for example, cyclopropyl, cyclohexyl, 1 ,2- dihydroxycyclopropyl, and the like.
  • cycloalkylalkyl refers to a moiety of the formula -R d R e where R d is an alkylene group and R e is a cycloalkyl group as defined herein.
  • exemplary cycloalkylalkyl groups include, but are not limited to, cyclopropylmethyl, cyclohexylpropyl, 3-cyclohexyl-2-methylpropyl, and the like.
  • the heterocyclyl ring can be optionally substituted with one or more, typically one, two, or three, substituents.
  • heterocyclyl groups include, but is not limited to, tetrahydropyranyl, piperidino, piperazino, morpholino and thiomorpholino, thiomorpholino-1 -oxide, thiomorpho lino- 1,1 -dioxide, and the like.
  • reacting are used interchangeably herein, and refer to adding or mixing two or more reagents under appropriate conditions to produce the indicated and/or the desired product. It should be appreciated that the reaction which produces the indicated and/or the desired product may not necessarily result directly from the combination of two reagents which were initially added, i.e., there may be one or more intermediates which are produced in the mixture which ultimately leads to the formation of the indicated and/or the desired product.
  • heteroaryl means a monovalent mono- or bicyclic aromatic moiety of 5 to 12 ring atoms containing one, two, or three ring heteroatoms selected from N, O, or S, the remaining ring atoms being C.
  • the heteroaryl ring can be optionally substituted with one or more substituents, typically one or two substituents.
  • heteroaryl includes, but is not limited to, pyridyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl, pyrrolyl, pyrazolyl, pyrimidinyl, benzofuranyl, isobenzofuranyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, indolyl, isoindolyl, benzoxazolyl, quinolyl, isoquinolyl, benzimidazolyl, benzisoxazolyl, benzothiophenyl, dibenzofuran, and benzodiazepin-2-one-5- yl, and the like.
  • steroid core structure refers to a moiety having the following general steroid ring system:
  • the steroid ring structure can be substituted and/or include one or more unsaturation.
  • said clinical condition comprises cancer such as, but not limited to, melanoma, breast cancer, prostate cancer, chronic lymphocytic leukemia, and/or lung cancer.
  • compositions comprising at least one compound disclosed herein, or an individual isomer, racemic or non-racemic mixture of isomers or a pharmaceutically acceptable salt or solvate thereof, together with at least one pharmaceutically acceptable carrier, and optionally other therapeutic and/or prophylactic ingredients.
  • the compounds are administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities.
  • Suitable dosage ranges are typically 1-500 mg daily, typically 1-100 mg daily, and often 1-30 mg daily, depending on numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, the indication towards which the administration is directed, and the preferences and experience of the medical practitioner involved.
  • One of ordinary skill in the art of treating such diseases is typically able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this application, to ascertain a therapeutically effective amount of the compounds of the invention.
  • compounds of the invention are administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal, or parenteral (including intramuscular, intraarterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
  • Typical manner of administration is generally oral using a convenient daily dosage regimen which can be adjusted according to the degree of affliction.
  • a compound or compounds of the invention, together with one or more conventional adjuvants, carriers, or diluents, can be placed into the form of pharmaceutical compositions and unit dosages.
  • the pharmaceutical compositions and unit dosage forms can be comprised of conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms can contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
  • compositions can be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of suppositories for rectal or vaginal administration; or in the form of sterile injectable solutions for parenteral use.
  • Formulations containing about one (1) milligram of active ingredient or, more broadly, about 0.01 to about one hundred (100) milligrams, per tablet, are accordingly suitable representative unit dosage forms.
  • the compounds of the invention can be formulated in a wide variety of oral administration dosage forms.
  • the pharmaceutical compositions and dosage forms can comprise a compound or compounds of the invention or pharmaceutically acceptable salts thereof as the active component.
  • the pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances which can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component.
  • the active component In tablets, the active component generally is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain from about one (1) to about seventy (70) percent of the active compound.
  • Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatine, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • the term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier, providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suitable for oral administration.
  • liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations which are intended to be converted shortly before use to liquid form
  • Emulsions can be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents, for example, such as lecithin, sorbitan monooleate, or acacia.
  • Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents.
  • Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents.
  • Solid form preparations include solutions, suspensions, and emulsions, and can contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • the compounds of the invention can also be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.
  • parenteral administration e.g., by injection, for example bolus injection or continuous infusion
  • the compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.
  • oily or nonaqueous carriers, diluents, solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and can contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
  • a suitable vehicle e.g., sterile, pyrogen-free water.
  • the compounds of the invention can be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch.
  • Ointments and creams can, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions can be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • Formulations suitable for topical administration in the mouth include lozenges comprising active agents in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatine and glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • the compounds of the invention can be formulated for administration as suppositories.
  • a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.
  • the compounds of the invention can also be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • the compounds of the invention can be formulated for nasal administration.
  • the solutions or suspensions are applied directly to the nasal cavity by conventional means, for example, with a dropper, pipette or spray.
  • the formulations can be provided in a single or multidose form. In the latter case of a dropper or pipette, this can be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this can be achieved for example by means of a metering atomizing spray pump.
  • the compounds of the invention can be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration.
  • the compound will generally have a small particle size for example of the order of five (5) microns or less. Such a particle size can be obtained by means known in the art, for example by
  • the active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC), for example, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, or carbon dioxide or other suitable gas.
  • CFC chlorofluorocarbon
  • the aerosol can conveniently also contain a surfactant such as lecithin.
  • the dose of drug can be controlled by a metered valve.
  • the active ingredients can be provided in a form of a dry powder, for example, a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP).
  • the powder carrier typically forms a gel in the nasal cavity.
  • the powder composition can be presented in unit dose form, for example, in capsules or cartridges of e.g., gelatine or blister packs from which the powder can be administered by
  • formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.
  • the compounds of the invention can be formulated in transdermal or subcutaneous drug delivery devices. These delivery systems are advantageous when sustained release of the compound is necessary or desired and when patient compliance with a treatment regimen is crucial.
  • Compounds in transdermal delivery systems are frequently attached to a skin-adhesive solid support.
  • the compound of interest can also be combined with a penetration enhancer, e.g., Azone (l-dodecylazacycloheptan-2-one).
  • Sustained release delivery systems can be inserted subcutaneously into the subdermal layer by surgery or injection.
  • the subdermal implants encapsulate the compound in a lipid soluble membrane, e.g., silicone rubber, or a
  • biodegradable polymer e.g., polylactic acid.
  • the pharmaceutical preparations are typically in unit dosage forms. In such form, the preparation is often subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • suitable pharmaceutical carriers and their formulations are described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa.
  • compositions which include therapeutically effective mounts of compounds of disclosed herein or pharmaceutically acceptable salts thereof or a prodrug thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously.
  • the compounds disclosed herein and pharmaceutically acceptable salts thereof are as described above.
  • the carrier(s), diluent(s), or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • a process for the preparation of a pharmaceutical formulation including admixing a compound disclosed herein, or a pharmaceutically acceptable salt thereof or a prodrug thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • compositions of this disclosure comprise a combination of a compound of the present disclosure and one or more additional therapeutic or prophylactic agent
  • both the compound and the additional agent are usually present at dosage levels of between about 10 to 150%, and more typically between about 10 and 80% of the dosage normally administered in a monotherapy regimen.
  • the Dynamic Character of the BCL2 Promoter i-Motif The i-motif structure was first characterized in 1993, but a potential role in transcriptional regulation has been proposed only recently. Unlike the G-quadruplexes found in promoters, which are stable under physiological conditions in single-stranded templates, i-motifs are far more dynamic and only stable at acidic pHs, because the cytosine-cytosine (C-C + ) base pair building block requires a hemiprotonated species ( Figure 1). Significantly, DNA, but not RNA, can form i- motifs because in DNA there is close contact between the deoxyribose sugars in the narrow groove that can give rise to favorable van der Waals energies.
  • i-motifs can form from duplex DNA and occur even in the absence of a G- quadruplex on the opposite strand. 6 Indeed, recently the i-motif and G-quadruplex were found to be mutually exclusive in the insulin-linked polymorphic region. Molecular crowding conditions mimicked by single-walled carbon nanotubes have been shown to induce telomeric i-motifs even at pH 8.0. Finally, it is believed that these species exist in a mixture of dynamic structures around their transitional states based on NMR, fluorescence resonance energy transfer (FRET), and differential scanning calorimetry studies.
  • FRET fluorescence resonance energy transfer
  • i-motifs found in the natural promoter sequences are classified as class I or class II i-motifs, with class I being more stable because of stabilizing interactions in their longer loop regions.
  • the BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif.
  • the i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species.
  • some compounds e.g., a pregnanol derivative and a class of piperidine derivatives
  • the BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, the present inventors have observed antagonism between the two classes of compounds both in solution and in cells. Some aspects of the invention are based on the discovery by the present inventors of the ability of some compounds to target i-motif structures in vitro and in vivo to modulate gene expression.
  • BCL2 PI promoter Directly upstream ( ⁇ 25 bases) from the BCL2 PI promoter is a GC-rich element known to form G-quadruplex and i-motif structures ( Figure 1).
  • Previous in vitro studies using synthetic oligomers have demonstrated that the BCL2 G-rich promoter element forms three different G-quadruplexes; the major one exhibits a mixed parallel/antiparallel structure.
  • the present inventors have previously demonstrated that the complementary C- rich sequence forms a stable i-motif structure with a high transitional pH of 6.6, likely due to stabilizing interactions in the central loop.
  • the present inventors have also discovered that the BCL2 i-motif is highly dynamic.
  • This dynamic equilibrium between the putative hairpin and the i-motif can be targeted through binding of a compound (e.g., cholestane) to the flexible hairpin or another compound (e.g., a pregnanol derivative) that binds to the central loop of the i-motif.
  • a compound e.g., cholestane
  • another compound e.g., a pregnanol derivative
  • BCL2 repression leads to chemosensitization of lymphoma to etoposide and significant reduction in tumor growth in a Granta-519 lymphoma model in SCID mice.
  • BCL2 i-Motif- and Hairpin-Interactive Compounds While G-quadruplexes in cellular DNA are known small-molecule targets, and the in vivo existence of these structures has been authenticated, no parallel role for the potential i-motifs on the opposite strand has yet been documented. Some aspects of the invention are based on the discovery by the present inventors of small molecules that can bind to the i-motif or an alternative unfolded form of this secondary structure.
  • a FRET high-throughput screening assay was used to identify small molecules from the NCI Diversity Set I (1,990 compounds) that interacted with the BCL2 i- motif promoter sequence. This screening assay led to the discovery of BCL2 i-motif- interactive small molecules that either stabilized or destabilized the structure as indicated by a decrease or increase in fluorescence intensity, respectively. A compound that decreased fluorescence by about 50% or increased fluorescence by at least 250% was considered a "hit.” This cut-off provided an overall i-motif-interactive hit rate of 0.7% (14/1,990), with 0.5%) for i-motif-destabilizing compounds (9/1,990) and 0.3%> for stabilizing compounds (5/1,990).
  • a cholestane derivative compound IMC-48 decreased the FRET signal by about 50%, while a pregnanol derivative carrying a benzoxazinyl substituent at C20, compound IMC-76, increased the BCL2 i-motif probe fluorescence by 270%> and were selected for further characterization. This led to a secondary screening of an additional 14 steroidal compounds from ChemDiv, all of which either increased the FRET intensity or had no effect (eight of the compounds that increased the FRET intensity are shown in Table 1). None of these steroidal compounds decreased the FRET signal like compound IMC-48. IMC-48 was selected for its BCL2 i-motif-stabilizing effect.
  • IMC-76 and IMC-48 interactions were specific for the BCL2 i-motif; no significant fluorescence increase or decrease was noted for other sequences (a BCL2 mutant sequence, the c-MYC and VEGF i-motif-forming sequences and the complementary BCL2 G-quadruplex sequence) or for the double-stranded region (BCL2 duplex).
  • IMC-76 and IMC-48 were used as contrasting compounds in parallel ex vivo and in vitro assays to demonstrate a correlation between the differential effects on FRET and the modulation of BCL2 transcription through small molecule interaction with the i-motif.
  • ID NMR Data The 1 D NMR of the imino region of the BCL2 i-motif and its equilibrating species are shown in Figure 2A. Significantly, at the transition pH 6.6, species corresponding to a duplex/hairpin and an i-motif were both clearly observed by 1H NMR ( Figure 2 A, traces 1 and 2). Two clear sets of imino proton signals are observed at around 13 and 15-16 ppm at pH 6.6 at 3 °C ( Figure 2A, trace 2).
  • the imino proton peaks at 15-16 ppm are characteristic of the hemiprotonated cytosine-cytosine base pairs in an i-motif and indicate the formation of a BCL2 i-motif, while the imino protons at 13 ppm are characteristic of Watson-Crick base pairs in a duplex or hairpin conformation.
  • the duplex/hairpin species appear to be in dynamic equilibrium with the BCL2 i-motif. Two different temperatures were used: a clearer signal of the hairpin conformation can be observed at low temperature, whereas i-motif signals are sharper at high temperature.
  • IMC-76 is a lipophilic molecule with limited functionality for H- bonding. Accordingly, it is believed that there is an entropic cost to keeping it as an isolated molecule in water related to the increased ordering of the water molecules around a species to which they cannot H-bond. Thus, just as in intercalative binding to duplex DNA, binding to the flexible hairpin can minimize the need for contact with water, providing the driving force to support the observed binding.
  • the finding ( Figure 2A, trace 7) that the complex has relatively well-resolved 1H NMR signals at -13 ppm suggests a relatively well-defined IMC- 76-flexible hairpin structure.
  • IMC-48 appears to redistribute the population from the flexible duplex to the i-motif by form stabilizing the i-motif population.
  • NMR competition titration experiments between IMC-48 and IMC-76 were carried out by either using a fixed equivalence of IMC-48 and increasing equivalence of IMC-76 (Figure 2C) or vice versa ( Figure 2D) at 25 °C and 3 °C.
  • the imino proton region shows a clear shift in the equilibrium from the i-motif to the flexible hairpin structure with the incremental addition of IMC-76 to the 2: 1 complex of IMC-48 and BCL2 DNA ( Figure 2C, traces 2-5). IMC-76 thus clearly shifts the equilibrium to the flexible duplex species, even in the presence of two equivalents of IMC-48.
  • IMC-76 and IMC-48 changed the molecular ellipticity in opposite directions; IMC-48 increased the molecular ellipticity, whereas IMC-76 decreased it.
  • IMC-48 stabilized the i-motif
  • IMC-76 bound to an alternative species that was in equilibrium with the i-motif.
  • the disruption of the i-motif structure by IMC-76 was further confirmed by bromine footprinting, which showed loss of protection of the C-C+ base pairs.
  • IMC-48 Since IMC-48 has a steroid core structure that appeared to bind to the i-motif, experiments were conducted to determine whether it was the steroid nucleus or a substituent that was responsible for the recognition and binding to the BCL2 i-motif.
  • One of the major structural differences between the IMC-76-type compounds and IMC-48 was the positively charged piperidine nucleus that is linked through an amide to the C3 position of the cholestane derivative.
  • Four additional compounds were acquired that mimicked this C3 substituent, and these compounds, shown below, were tested alongside IMC-48 initially in a FRET-based assay.
  • BCL2 i-motif-interactive compounds were assessed in three lymphoma cell lines that differentially express BCL2: (1) Epstein Barr Virus (EBV) negative parental Burkitt's lymphoma cells (BJAB), which express little to no BCL2; (2) EBV- infected parental cells (B95.8), which express significantly higher levels of BCL2 and display apoptotic resistance to etoposide; and (3) EBV-positive GRANTA-519 mantle cells, which express similar levels of BCL2 to B95.8 cells ( Figure 3 A). Cells were treated with increasing concentrations of IMC-48 and IMC-76 for 24 h based on the IC 50 values to avoid cytotoxicity ( Figure 5 and Table 2).
  • EBV Epstein Barr Virus
  • BJAB Burkitt's lymphoma cells
  • B95.8 EBV- infected parental cells
  • EBV-positive GRANTA-519 mantle cells which express similar levels of BCL2 to B95.8 cells
  • BCL2 expression in BJAB cells was unaffected by IMC-76 ( Figure 3B, lower) since the basal levels of BCL2 expression are negligible. Downregulation of BCL2 expression was also observed at the protein level ( Figure 6).
  • the BCL2 protein expression was evaluated by western blot analysis, and a representative blot using lysate from one mouse within each group showing a decrease in BCL2 is shown in Figure 4A, right.
  • a follow-up combination study with the GRANTA-519 xenograft SCID mice (N 12) revealed that co-treatment of 10 mg/kg IMC-48 and etoposide significantly reduced the tumor burden when compared to etoposide alone ( Figure 4B, left).
  • a transcriptional factor that recognizes and binds to the BCL2 i-motif to activate transcription.
  • the molecular basis for the recognition of the i-motif by hnRNP LL was determined, and the present inventors have discovered that the protein unfolds the i-motif structure to form a stable single-stranded complex.
  • IMC-48 and IMC-76 have opposite, antagonistic effects on the formation of the hnR P LL-i-motif complex as well as on the transcription factor occupancy at the BCL2 promoter.
  • the i-motif acts as a molecular switch that controls gene expression and that small molecules, including compounds of the invention, that target the dynamic equilibrium of the i-motif and the flexible hairpin can differentially modulate gene expression.
  • DNA secondary structures serve as switches to turn gene transcription on or off.
  • the present inventors have discovered small molecules that bound to different topological forms of the C-rich strand of the BCL2 cis-regulatory element and either repressed or activated transcription.
  • Compounds such as IMC-48
  • the compounds such as IMC-76
  • Antagonism between the two groups of compounds was found to occur with the DNA species in solution as well as within a cellular system. On the basis of these results, it was believed that there is/are transcriptional factor(s) that would similarly bind to the two different DNA structures, thereby mimicking the effect of these two groups of compounds on BCL2 gene expression.
  • hnRNP LL is a transcriptional factor that recognizes the BCL2 i-motif and subsequently unfolds it to activate transcription. It should be noted that hnRNP LL belongs to the same protein family as hnRNP K, which previously was shown to activate MYC transcription by binding to the C-rich strand of the MYC promoter. Following the identification of hnRNP LL as an activating transcriptional factor for BCL2, it was shown by the present inventors that compounds that bind exclusively or selectively to one or the other of the two equilibrating species of the BCL2 C-rich strand exert their activity by modulating the amount of the i-motif available for binding to hnRNP LL.
  • G-quadrup lex-binding agents can interfere with protein- DNA complex formation, potentially modulating gene expression.
  • Experiments were conducted to identify nuclear proteins that could bind to the i-motif or an unfolded form and modulate BCL2 transcriptional. Since the i-motif is highly dynamic, any identified i-motif binding protein may take advantage of this property and form a stable DNA complex by i- motif remodeling. The C-rich strand that gives rise to the folded i-motif has features more commonly associated with secondary RNA structures than DNA, thus RNA-binding proteins were considered.
  • Candidates included RNA recognition proteins belonging to the hnRNP class normally associated with RNA splicing. Although not as yet reported to bind to an i- motif structure, an example is hnRNP K, which binds to the CT element of the MYC promoter to activate transcription.
  • Nuclear proteins from HeLa nuclear extract that putatively bind to the BCL2 i- motif were purified using a biotinylated oligomer-streptavidin bead complex pull-down assay and identified by liquid LC/MS/MS sequencing.
  • Two biotinylated oligomer-bead complexes were used consisting of either the wild-type BCL2 i-motif-forming sequence or a mutant oligomer (which cannot form a stable i-motif) for nonspecific protein binding.
  • Proteins that bound uniquely to the BCL2 i-motif-forming promoter element were classified into functional groups: (1) transcription, (2) translation or protein- folding, (3) energy metabolism or other enzymatic processes, and (4) cell adhesion or migration functions, mostly related to the cytoskeleton (Tables 3 to 5). Of interest were proteins having documented function related to transcription, (Table 3) particularly hnRNP LL.
  • hnRNP L is a pre- mRNA splicing factor, which binds to and stabilizes BCL2 mRNA.
  • the hnRNP LL protein is a paralog of hnRNP L, shows tissue-specific distribution, and activates T-cells by shifting transcriptomes for cellular proliferation and inhibition of cell death.
  • HMG-I Isoform HMG-I of high-mobility group protein HMGI/HMG- Y IPI00179700 hnRNP UL2 Heterogeneous nuclear ribonucleoprotein U-like protein 2 IPI00456887 hnRNP LL Isoform 1 of heterogeneous nuclear ribonucleoprotein L-like IPI00103247
  • HMGN1 Nucleosome-binding protein 1 IPI00006157
  • EIF3A Eukaryotic translation initiation factor 3 subunit A IPI00029012
  • NMT-1 Isoform short of glycylpeptide N-tetradecanoyltransferase 1 IPI00218830
  • NCL-like cDNA FLJ45706 fis highly similar to Nucleolin IPI00444262 hnR P A2/B1 Putative uncharacterized protein FJNRNP A2B1 IPI00386854 hnRNP Al Isoform Al-B of heterogeneous nuclear ribonucleoprotein Al IPI00215965 hnRNP R Heterogeneous nuclear ribonucleoprotein R IPI00012074 hnRNP G Heterogeneous nuclear ribonucleoprotein G IPI00304692 hnRNP A3 Isoform 1 of heterogeneous nuclear ribonucleoprotein A3 IPI00419373
  • ILF-3 Isoform 5 interleukin enhancer-binding factor 3 IPI00219330
  • HSC71 Isoform 1 of heat shock cognate 71 kDa protein IPI00003865
  • PPIase B Peptidyl-prolyl cis-trans isomerase B IPI00646304
  • BCL2 i-Motif Recognition by hnRNP LL The hnRNP LL protein shares 57% sequence identity to hnRNP L. Both proteins have four RNA recognition motifs (RRMs) and at least two are required for stable binding to single-stranded RNA or DNA. Two consensus sequences for binding these RRMs are found in the BCL2 i-motif, and both are located in the lateral loops. To determine the importance of these loops in comparison to the central loop, cold mutant Py39 sequences were designed having one or more of these loops mutated, but still maintaining the basic i-motif core structure.
  • RRMs RNA recognition motifs
  • hnRNP LL recognize the mixed cytosine/guanine sequences in the lateral loops by binding to one or both of the lateral loops (the 5 ' lateral loop is the favored one). Then, after subsequent protein-facilitated i-motif unfolding, hnRNP LL binds more stably to an unfolded i-motif species not present initially.
  • hnRNP LL The i-motif unfolding activity of hnRNP LL was further confirmed by a quencher-based FRET assay.
  • hnRNP LL increased the fluorescence intensity by 1.8-fold at pH 6.5 where the i-motif is expected to be initially present, but had little effect at pH 7.9 where the i-motif is absent.
  • hnRNP LL selectively increased the fluorescence signal of wild-type sequence compared to lateral loop mutant (Mut5', 3'L) at pH 6.5. This result strongly suggests that the binding and associated unfolding activity of hnRNP LL is restricted to the i-motif structure with wild-type sequence in lateral loop.
  • hnRNP LL binding to two similar consensus sequences in the C-rich strand of the BCL2 promoter that results in transcriptional activation is quite analogous to hnRNP K binding to the CT elements in MYC NHE IIL .
  • the hnRNP K protein contains three KH domains that are spaced apart in a similar manner to hnRNP LL, but recognize TCCC sequences.
  • TCCC elements are found in the lateral loops of the MYC i-motif and are spaced the same distance apart in the unfolded structure as those found in the BCL2 i- motif.
  • hnRNP K and hnRNP LL may have similar roles in transcriptional activation of MYC and BCL2 : they recognize similar single-stranded elements in the lateral loops of their respective i-motifs, and both presumably remodel the i-motif to form a thermodynamically stable species prior to transcriptional activation.
  • Compound IMC-76 can change the dynamic chemical populations of equilibrating C-rich strand species in solution by sequestering the flexible hairpin.
  • the RRMs of hnRNP LL require the presence of the CGCCC and CCCGC sequences in the lateral loops of the i-motif for optimum binding and subsequent unfolding leading to transcriptional activation. Taken together, this suggests a competition between IMC-76 and hnRNP LL for binding to the equilibrating populations of flexible hairpin and i-motif. Binding of IMC-76 to the flexible hairpin increases the population of this species and depletes the population of the hnRNP LL-bound i-motif species.
  • IMC-76 decreases the i-motif population in the promoter element and thus reduces hnRNP LL promoter occupancy.
  • compound IMC-48 like hnRNP LL, binds exclusively to the BCL2 i-motif, thus IMC-48 increases the i- motif population and thereby increases the amount of hnRNP LL-bound i-motif species— assuming that hnRNP LL binds to the i-motif tightly enough to displace IMC-48— and increases the promoter occupancy in cells.
  • EMSA knockdown of hnRNP LL, and ChIP analysis was also performed.
  • IMC-48 As the concentration of IMC-48 increased, there was a depletion of species 1 and species 2, and an increased band intensity of the hnRNP -BCL2 i-motif complex. This indicates that the increase of i-motif population by IMC-48 facilitates the binding of hnRNP LL to the i-motif structure.
  • MCF-7 and BJAB cells were treated with IMC-76 and IMC-48 (at 0.5 and 2 ⁇ ) for 24 h, respectively.
  • Quantification of immunoprecipitated DNA was performed by SYBR green I qPCR using two specific sets of primers, amplifying either the closest upstream region (-103 to -3 base pairs) or a far upstream region (>3000 base pairs) from the i-motif/G-quadruplex-forming site of the PI promoter, the latter serving as a negative control for normalization.
  • IMC-76 decreased the occupancy of both Spl and hnRNP LL bound to the BCL2 PI promoter region in a concentration-dependent manner in MCF-7 cells.
  • IMC-48 increased the promoter occupancy of both Spl and hnRNP LL in BJAB cells.
  • qPCR was carried out.
  • IMC-48 on the transcription level of Spl and hnRNP LL was tested with BJAB cells.
  • immunoprecipitation (IP) experiments were carried out for both Spl and hnR P LL to verify antibody specificity.
  • IMC-48 and IMC-76 are antagonistic in redistribution of the two populations of DNA species in solution using 1D-NMR studies as well as in cellular studies by following the chemosensitization to cyclophosphamide.
  • 1D-NMR studies As well as in cellular studies by following the chemosensitization to cyclophosphamide an experiment was carried out in MCF-7 cells in which Spl and hnRNP LL were first depleted from the promoter element by treatment with IMC-76. After 24 h, the cells were treated with IMC-48, which was expected to reverse these effects relative to the control in which only IMC-76 has been previously added.
  • the i-motif is perhaps the most dynamic at pH levels that are either slightly acidic or even close to neutral. Because the i-motif is formed from hemiprotonated C-CH+ base pairs which have a ⁇ ⁇ of 4.58 for the N3 of cytosine, their existence in cells has not been generally anticipated. However, an important contributor to their increased stability is favorable van der Waals energies, due to close contacts between deoxyribose sugars in the narrow groove of the tetrad and this is dependent upon the precise topology of
  • phosphodiester backbone with intercalation of C-CH + pairs Significantly i-motifs in RNA cannot be formed, even at low pH because of the steric hindrance of the 2'-hydroxyl group. Since the topology of the phosphodiester backbone appears to be critical in stabilization of the i-motif through sugar-sugar interaction, conditions such as molecular crowding, negative superhelicity and loop constraints may play important roles if they influence these
  • promoter i-motifs have dynamic properties more like R A secondary structures than what are typically associated with DNA.
  • Both small molecules e.g., IMC-76 and IMC-48
  • a transcriptional factor hnRNP LL
  • Both small molecules e.g., IMC-76 and IMC-48
  • hnRNP LL transcriptional factor
  • Both small molecules e.g., IMC-76 and IMC-48
  • hnRNP LL transcriptional factor
  • the competition between these ligand- or protein- associated dynamic states has functional consequences, leading to gene expression modulation. This is analogous to metabolite-sensing riboswitches that regulate gene expression in response to small molecules by causing a redistribution of the conformational states with functional consequences.
  • the underlying common feature of the BCL2 i-motif and the riboswitch is the ability of ligands and proteins to take advantage of the intrinsic dynamic chemical behavior of DNA or RNA.
  • hnRNP L The recognition and subsequent stable binding of hnRNP LL to the BCL2 i- motif was more complex.
  • the hnRNP LL protein and its paralog hnRNP L share a 58% overall amino acid identity and contain four classical RRMs that are highly conserved.
  • the overall arrangement of the RRMs in hnRNP L and hnRNP LL are similar, such that in both cases they are separated by linkers of different lengths so they can recognize either adjacent domains or ones spaced further apart.
  • a combination of at least two RRMs is required for the high-affinity binding of hnRNP L to RNA.
  • hnRNP L binding site consists of 21 nucleotides in mRNA 3 ' UTR approximately equivalent to the 23 combined nucleotides contained in the two lateral loops and the linker region recognized by hnRNP LL.
  • the conformational change in the VEGF 3 ' UTR is directed by two different signals, hnRNP L and INF-y-activated inhibitor of translational complex, which bind to two different RNA conformers in a mutually exclusive manner, just as hnRNP LL and IMC-76 bind to the i-motif and flexible hairpin in the BCL2 promoter.
  • the hnRNP LL protein binds with high affinity to the BCL2 i-motif (20-70 pM) and siRNA knockdown significantly decreased BCL2 expression. Recognition of the i- motif is through the 5 ' and 3 ' lateral loops, but subsequent unfolding of the i-motif is presumably required before a stable complex is formed. It is likely that both lateral loops are initially recognized by adjacent RRMs before subsequent hnRNP LL-driven changes in the interhelical conformation, so that the 5 ' and 3 ' lateral loops are driven apart to bind to the RRMs spaced further apart, ( Figure 13, A-C and D).
  • IMC-76 In cells the competition for the BCL2 i- motif species by IMC-76, which depletes this population, reduces the amount of hnRNP LL bound to the BCL2 promoter, as determined by ChIP analysis, whereas IMC-48 produces the opposite effect by constraining the i-motif structure.
  • hnRNP K activates MYC transcription and binds to the CT elements in the promoter, probably by a similar mechanism to hnRNP LL. At least two other factors may be important in the mechanism for transcriptional activation by hnRNP LL.
  • DDX21 an RNA helicase, also bound to the i-motif (Table 3) or to an associated protein, and this may be important in facilitating i-motif unfolding to activate transcription.
  • BCL2 mediated by compounds related to IMC-48 provides a means to protect against neurodegenerative diseases, such as those found in CNS disorders. This brings the i-motif into focus as an alternative structure to the G-quadruplex in promoter elements as a therapeutic target.
  • Etoposide was purchased from Sigma-Aldrich (St. Louis, MO). Abbott laboratories (Abbott Park, IL) kindly provided ABT-737. All compounds were dissolved in 100% DMSO to obtain a 10 mM stock concentration based on the molecular weight of each compound. Stock compounds were then diluted to working concentrations with deionized water or tissue culture medium.
  • FRET Assay FRET probes were synthesized by Biosearch (Novato, CA) with a 5 ' -end FAM-fluorophore and a 3 ' -end black hole quencher. Probes were prepared and fluorescence was measured. For the high-throughput screen, the BCL2 i-motif probe (1 ⁇ ) was incubated with compounds from the NCI diversity set (5 ⁇ ) at pH 5.8 (50 mM Na cacodylate buffer). Samples were prepared in single wells of the 96-well plate, according to the NCI predetermined plate set-up.
  • the BCL2 i-motif-forming oligomers were synthesized by Eurofms MWG Operon (Huntsville, AL) or Biosearch Technologies (Petaluma, CA).
  • the BCL2 wild-type oligomer was diluted to a 5 ⁇ strand concentration and incubated with 1 and 2 equivalents of IMC-76 and IMC- 48 in 10 mM Na cacodylate buffer (pH6.3) and 50 mM buffer (pH6.6), respectively.
  • the BCL2 mutants (Mut5 ' ,3 ' L and MutCL) at 50 mM Na cacodylate (pH6.3) were heated at 95 °C for 5 min and slowly cooled to room temperature.
  • IMC-48 and IMC-42 were incubated with the oligomers for 20 min prior to CD analysis.
  • the instrument was set to gather spectral data over a wavelength range of 230-330 nm with a scanning speed of 100 nm/min and a response time of 1 s. All spectra were recorded in triplicate, averaged, baseline-corrected for signal contributions from buffers, and smoothed out. Molar ellipticities for melting curves were recorded at 286 nm (the ⁇ of the maximum molar ellipticity).
  • CD spectra were baseline corrected by subtracting a buffer alone or a buffer with compound.
  • the final NMR samples were prepared in 10%/90% D 2 O/H 2 O solution at pH 6.0 and 6.6. The concentration of DNA samples was 0.3 mM.
  • the stock solutions of IMC-76 and IMC-48 were dissolved in d 6 -DMSO.
  • One-dimensional 1H NMR titration experiments were performed on a Bruker DRX-600 MHz spectrometer at temperatures of 25 °C and 3 °C. The WATERGATE technique was used to suppress the water signal in the 1H NMR experiment.
  • Naphthodeoxyuridine Fluorescence Assay The fluorescent thymidine substitute (NdU phosphoramidite) was prepared and incorporated into oligonucleotides. Strand concentrations were calculated and fluorescence measurements were conducted. The extinction coefficient used for the NdU was 49,800 M _1 cm -1 . The extinction coefficient for each oligomer (T20, T21, T24, and T39) was 328,680 M _1 cm -1 . Each probe was placed in a 50 mM Na cacodylate buffer (pH 6.3) at a strand concentration of 10 ⁇ in the absence or presence of compound at increasing molar concentration equivalents.
  • the samples were incubated at 95 °C for 5 min and allowed to cool to room temperature (25 °C) to allow for i- motif formation.
  • the excitation and emission wavelengths were set at 250 nm and 440 nm, respectively. Endpoint fluorescence or quenching was plotted as the average percent change in fluorescence relative to probe alone of the triplicate wells after correction for background.
  • MCF-7, MDA-MB-231, and GRANTA-519 cell lines were purchased from the American Type Culture Collection (Manassas, VA).
  • MCF-7 tamoxifen-resistant (MCF-7/TAMR) cell line was obtained from the University of Arizona Experimental Mouse Shared Service (Tucson, AZ). All cell lines were cultured in 10% FBS, 5% penicillin/streptomycin- supplemented RPMI. MCF-7/TAMR cells were also cultured in the presence of tamoxifen. Cells were assessed for viability (>90%>) by trypan blue exclusion prior to use for experimental purposes. All cell culture experiments were conducted at 24 h unless otherwise stated.
  • the University of Arizona Genetics Core using a forensic-style 15 autosomal STR loci including 13 CODIS DNA markers and Amelogenin, authenticated the BJAB and B95.8 cell lines.
  • the GRANTA-519 cell lines were authenticated using a STR analysis including nine autosomal STR loci, Amelogenin, and a mouse-specific locus.
  • TaqMan probes were used for BCL2 (HsOO 153350 or Hs00608023) and GAPDH (Hs02758991) PCR amplification. Samples analyzed with ⁇ -actin as a reference gene. For IMC-42 (0.5, 2 ⁇ ), real-time PCR was performed using Rotor-Gene Q (Qiagen).
  • Protein bands were resolved on a precast 10%> sodium dodecyl sulfate polyacrylamide gel (BioRad, Hercules, CA) from 40 ⁇ g total protein. Protein was then transferred to a polyvinylidene difluoride membrane using the iBlot system for the recommended voltage and time (Invitrogen, Grand Island, NY). Membranes were incubated overnight at 4 °C with monoclonal antibodies targeting BCL-2 (Cell Signaling, Dancers, MA) and ⁇ -actin (Abeam, Cambridge, MA) which were used at a dilution of 1 : 1000 and 1 :5000, respectively.
  • BCL-2 Cell Signaling, Dancers, MA
  • ⁇ -actin Abeam, Cambridge, MA
  • MCF-7, MCF-7/TAMR, MDA-MB-231, BJAB, B95.8, and GRANTA-519 were determined by the MTS colorimetric assay as per the manufacturer's specifications
  • Caspase-3 Activity Assay Caspase-3 activity was evaluated using the
  • SC tumors were measured for tumor volume estimation (cm 3 or mm 3 ) in accordance with the formula a 2 x b/2, where a equals the smallest diameter and b is the largest diameter. Tumors were only allowed to reach 2000 mm 3 .
  • the Grubbs' or maximum normed residual test was used to detect outliers in each treatment group. One outlier was found and excluded from statistical analyses. Significant differences in AUC values were determined using one-way ANOVA.
  • i-Motif Protein Binding Purification Assay All of the following incubations, washes, and centrifugations (1 min at 500 g) were performed at 4 °C.
  • the biotinylated BCL2 i-motif wild-type and mutant oligomers (4 ⁇ g each) were conjugated to washed streptavidin beads in separate 1.5 mL Eppendorf tubes in binding Buffer B (1 mM DTT, 25 mM Tris HCl [pH 7.6], 50 mM NaCl, 0.5 mM MgCl 2 , 1 mM EDTA, 10% glycerol) plus l protease inhibitor cocktail overnight, rotating.
  • the mutant oligomer-conjugated beads were incubated with 500 ⁇ g HeLa extract for 3 h, rotating. The beads were centrifuged and supernatant was transferred to the wild-type oligomer-conjugated beads and incubated for 3 h, rotating.
  • the mutant oligomer-conjugated beads were incubated with 500 ⁇ g HeLa extract for 3 h, rotating. The beads were centrifuged and supernatant was transferred to the wild-type oligomer-conjugated beads and incubated for 3 h, rotating.
  • the mutant oligomer-conjugated beads were incubated with 500 ⁇ g HeLa extract for 3 h, rotating. The beads were centrifuged and supernatant was transferred to the wild-type oligomer-conjugated beads and incubated for 3 h, rotating.
  • the mutant oligomer-conjugated beads were incubated with 500 ⁇ g HeLa extract for 3 h, rotating. The beads were centr
  • oligomer/bead/HeLa extract complex was washed in Buffer B, and supernatant from each wash was transferred to the wild-type oligomer. Proteins were eluted off the mutant oligomer-conjugated beads with successive washes of a NaCl gradient (0.1-2 M) in Buffer B, and each supernatant was collected and combined. The wild-type oligomer/bead/HeLa nuclear extract complex was subjected to the same procedure of washing and elution as described for the mutant oligomer complex. The eluted proteins were processed by the BI05 Proteomics Core Facility (BI05 Institute, Arlington, AZ). The two protein samples were subjected to SDS PAGE and visualized by Coomassie and silver staining. Prominent bands were excised from the gel and analyzed for protein identification by LC/MS/MS.
  • hnRNP LL The cDNA of hnRNP LL was purchased from Open Biosystems (ThermoScientific) and subsequently cloned into the pET28a protein expression vector (Novagen). After sequencing analysis to confirm the pET28a-hnRNP LL, this expression construct was transformed into Rosetta-gamiTM B (DE3) pLysS cells (Novagen). The expression of hnRNP LL was induced by 0.1 mM IPTG
  • the resin was washed by washing Buffer A (50 mM NaH 2 P0 4 with 0.4x protease inhibitor cocktail) and B (50 mM NaH 2 P0 4 [pH 8.0] and 100 m NaCl with 0.1 * protease inhibitor cocktail) sequentially, and elution buffer (50 mM NaH 2 P0 4 [pH 8.0], 300 mM NaCl with l protease inhibitor cocktail) was used to separate hnRNP LL from resin.
  • Buffer A 50 mM NaH 2 P0 4 with 0.4x protease inhibitor cocktail
  • B 50 mM NaH 2 P0 4 [pH 8.0] and 100 m NaCl with 0.1 * protease inhibitor cocktail
  • elution buffer 50 mM NaH 2 P0 4 [pH 8.0], 300 mM NaCl with l protease inhibitor cocktail
  • Purified hnRNP LL was subjected to buffer exchange into a protein stock buffer with 20 mM HEPES-NaOH [pH 7.4], 100 mM KC1, 10% Glycerol, 2 mM DTT, and 0.1% NP-40) using a centricon
  • EMS A All oligomers for these experiments were purchased from Eurofms MWG Operon and PAGE -purified. Concentrations of purified oligomers were determined using the Lambert-Beer equation with molecular extinction coefficients (M _1 cm 1 ) as follows: Py39WT, 292,338; Py39MutT, 319,216; Pu39WT, 398,551. The wild-type BCL-2 i- motif (Py39WT) oligomer was end-labeled with [ ⁇ - 32 ⁇ ]- ⁇ .
  • Biacore T100 optical biosensor with CM5 sensor chips (GE Healthcare, Piscataway NJ). N- hydroxysuccinimide, l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, and ethanolamine, 1 M (pH 8.5), were purchased from GE Healthcare. Biotinylated oligomers with wild-type and mutant sequences were purchased from Eurofms MWG Operon.
  • biotinylated oligomers were resuspended in 10 mM Tris (pH 8.0), 1 mM
  • EDTA at 100 ⁇ , then diluted to 1 ⁇ in 20 mM HEPES (pH 7.9), 100 mM KC1, 2 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 1 ⁇ g/ ⁇ L BSA, 0.1% Tween 20, and 10% glycerol.
  • the diluted oligomers were heated at 95 °C for 5 min, cooled to rt, and centrifuged at 16K x g for 10 min at rt. The supernatant was diluted to 1 iiM in the same buffer and injected over the active surface at 10 ⁇ / ⁇ until 5 RU was captured.
  • hnRNP LL was diluted into running buffer (20 mM HEPES for pH 7.9 and pH
  • Raw data were reference subtracted and buffer blanks were subtracted (double referencing).
  • Data were fit to a 1 : 1 binding model using a global fit algorithm (Biacore T100 Evaluation Software) to obtain the kinetic parameters ka, kd, and K D .
  • the i-motif-forming oligomers were synthesized by Euro fins MWG Operon. Py39WT and Py39MutT were diluted to 5 ⁇ with a buffer (50 mM MES [pH 6.5], 100 mM KC1, 2 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 1 ⁇ g/ ⁇ L BSA, 0.1% Tween 20, and 10 % glycerol).
  • a buffer 50 mM MES [pH 6.5], 100 mM KC1, 2 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 1 ⁇ g/ ⁇ L BSA, 0.1% Tween 20, and 10 % glycerol.
  • Recombinant hnRNP LL was diluted by protein stock buffer to desired concentrations to maintain consistent buffer conditions in each sample. Oligomers and hnRNP LL were incubated for 5 min at room temperature. CD spectra were baseline corrected by subtracting a buffer alone or a buffer with protein
  • Bromine Footprinting For the Br 2 footprinting of the BCL-2 i-motif and hnRNP LL complex, recombinant hnRNP LL was incubated with end-labeled Py39WT in a buffer (50 mM MES [pH 6.5], 4 mM MgCl 2 , 100 mM KC1, 1 mM DTT, 1 ⁇ g/ ⁇ L BSA, 0.1% Tween 20, 10%> glycerol, and 0.02 ⁇ g/ ⁇ L poly [dl-dC]) for 5 min at room temperature.
  • a buffer 50 mM MES [pH 6.5], 4 mM MgCl 2 , 100 mM KC1, 1 mM DTT, 1 ⁇ g/ ⁇ L BSA, 0.1% Tween 20, 10%> glycerol, and 0.02 ⁇ g/ ⁇ L poly [dl-dC]
  • Bromination was conducted by addition of 0.1 mM bromine for 30 min at room temperature, and subsequently a phenol/chloroform solution was added to interrupt the bromination and remove the protein. Brominated oligomer was subjected to EtOH precipitation. The pellet was washed with 80% EtOH and treated with 10% piperidine at 93 °C for 15 min to induce the bromination-specific DNA cleavage. Cleaved product was washed with water and visualized by a 20%> sequencing gel with 7M urea.
  • siRNA Knockdown Assay siRNA (ID: SASI HsOl 00171042 and
  • the cDNA was synthesized by a reverse-transcription kit (Qiagen or Takara with gDNA remover) and used as templates for qPCR with TaqMan probes for hnRNP LL (Hs00293181_ml , FAM-labeled), BCL-2 (HS00608023_ml, FAM-labeled), and GAPDH (Hs02758991_gl, VIC-labeled) (ABI).
  • the C t values were obtained by Rotor-Gene Q (Qiagen) to analyze the relative quantity of hnRNP LL and BCL-2 mRNA compared to GAPDH as an internal control.
  • Promoter Assay The pGL3-BCL-2 wild-type construct was prepared using the BCL-2 promoter region from -35 to +614, which includes the i-motif-starting site. The sequence was inserted into the pGL3-basic vector at the Kpnl and Nhel restriction sites. The pGL3-Mut5 ' ,3 ' L and pGL3-MutCL constructs were generated by site-directed mutagenesis. The sequences of each construct were confirmed by sequencing analysis.
  • MCF-7 cells (1.5 x 10 4 ) were transfected with 500 ng of pGL3 construct, 10 ng of pRL-TK, and 50 nM of negative control or hnRNP LL siRNA by Fugene HD transfection reagent and incubated for 72 h.
  • Cells were lysed by passive lysis buffer (Promega), and then supernatants were subjected to dual-lucif erase assays (Promega) using an FBI 2 luminometer (Berthold detection system). Data was normalized to the ratio of firefly to renilla luciferase of siRNA- treated sample and to siRNA-untreated control.
  • ChIP Assay Both MCF-7 cells (5 10 5 ) and BJAB cells (1 10 6 ) were cultured overnight and then treated for an additional 24 h with 0.5 of IMC-76 or 2 ⁇ IMC- 48. Treatment with DMSO served as the control. To determine the antagonistic effect of two compounds, MCF-7 cells (3-4 x 10 5 ) were treated with DMSO or 2 ⁇ of IMC-76 for 24 h. The next day, DMSO-treated cells were administered with DMSO or IMC-76, and IMC-76- treated cells were administered with 2 or 4 ⁇ of IMC-48 with fresh media for 24 h. The composition of the buffers used for this ChIP assay is the same as those of the EZ ChIP kit (Millipore).
  • MCF-7 cells and BJAB cells were lysed with 1% SDS buffer and sonicated to fragment chromosomal DNA into -500 base pairs for 15 and 45 cycles, respectively. Sheared chromosomal DNA was diluted with ChIP dilution buffer and precleaned with Protein G- coupled Dynabeads (Invitrogen) for 2 h at 4 °C.
  • the DNA was purified using a PCR purification kit (Qiagen), and SYBR Green I qPCR analysis was performed with Rotor-Gene Q (Qiagen) to determine relative quantity of DNA using primers to specifically amplify the -3— 103 bp from the BCL-2 i-motif-forming region within the promoter.
  • An upstream region (— 3456 base pairs) from this i-motif- forming region was also amplified to serve as a negative control for normalization.
  • Melting analysis of PCR product showed only one detectable T m (data now shown), and double normalizations were performed to obtain data (2 ⁇ AACt ).
  • AC t values were calculated by subtracting C t values of negative region (C t -C t neg) and then ⁇ values were obtained by normalizing to AC t of input (AC t -AC t input).
  • MCF-7 cells 1.5 10 5
  • BJAB cells 3 10 5
  • Total RNA extraction, cDNA synthesis, and qPCR were performed using the gene-specific TaqMan probes.
  • the specificity and IP-quality of Spl and hnRNP LL antibodies are demonstrated by the manufacturer and further verified by
  • proteins were transferred to PVDF membrane in TBS buffer with 20% MeOH. After blocking the membrane with 2% BSA/2% nonfat milk in TBS-T (0.1% Tween 20) for 1 h, Spl antibody with 1 : 1000 dilution and hnRNP LL antibody withl :300 in 1% BSA/TBS-T were treated overnight at 4 °C.
  • As a secondary antibody goat anti-rabbit IgG (H+L) Dylight 680 was diluted into 1 : 10,000 in 1% BSA/ TBS-T and incubated for 1 h at rt. LI-COR was used to detect the bands.

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Abstract

La présente invention concerne un procédé pour traiter un trouble pathologique associé à la surexpression du gène BCL2. En particulier, la présente invention concerne un procédé pour traiter un trouble pathologique associé à la surexpression du gène BCL2 par administration d'un composé qui réduit la structure du motif i du gène BCL2.
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WO2002097053A2 (fr) * 2001-05-30 2002-12-05 The Regents Of The University Of Michigan Petits antagonistes moleculaires de proteines de la famille bcl2
FR2964323A1 (fr) * 2010-09-08 2012-03-09 Jean Pierre Raynaud Utilisation de la testosterone chez un patient en deficit androgenique et atteint d'un cancer de la prostate

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FR2964323A1 (fr) * 2010-09-08 2012-03-09 Jean Pierre Raynaud Utilisation de la testosterone chez un patient en deficit androgenique et atteint d'un cancer de la prostate

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