WO2015123618A1 - Method for increasing bcl2 gene expression - Google Patents

Method for increasing bcl2 gene expression Download PDF

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WO2015123618A1
WO2015123618A1 PCT/US2015/015982 US2015015982W WO2015123618A1 WO 2015123618 A1 WO2015123618 A1 WO 2015123618A1 US 2015015982 W US2015015982 W US 2015015982W WO 2015123618 A1 WO2015123618 A1 WO 2015123618A1
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bcl2
imc
motif
hnrnp
compound
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PCT/US2015/015982
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French (fr)
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Laurence Hurley
Samantha KENDRICK
Vijay Gokhale
Hyunjin Kang
Danzhou Yang
Prashansa AGRAWAL
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Arizona Board Of Regents For The University Of Arizona
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Publication of WO2015123618A1 publication Critical patent/WO2015123618A1/en

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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 underexpression of BCL2 gene.
  • the present invention relates to treating a clinical condition associated with underexpression of BCL2 gene by administering a compound that increases 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 method for therapeutic intervention based on shifting the equilibrium populations of BCL2 gene conformational isomers. In one particular embodiment, the method increases formation of the i-motif conformation of BCL2 gene compared to the flexible hairpin conformation of BCL2 gene.
  • the amount of relative increase in the i- motif (relative to the amount of the i-motif 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 DNATMC-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 underexpression of BCL2 gene. Such methods include administering to a subject in need of such a treatment a compound that increases i-motif structure of BCL2 gene, thereby increasing the expression of BCL2 gene in the subject. Methods of the invention increases 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.
  • Exemplary clinical conditions that can be treated with methods of the invention include neurodegenerative diseases as well as other clinical conditions that are caused by underexpression of BCL2 gene.
  • methods of the invention can be used to treat amyotrophic lateral sclerosis (ALS) or a neurodegenerative disease such as schizophrenia, Alzheimer's disease, Huntington disease and other neurodegenerative diseases caused by underexpression of BCL2 gene.
  • ALS amyotrophic lateral sclerosis
  • a neurodegenerative disease such as schizophrenia, Alzheimer's disease, Huntington disease and other neurodegenerative diseases caused by underexpression of BCL2 gene.
  • the compound decreases 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 more often at least 90% when the ratio of said compound to BCL2 DNA is about 4: 1.
  • the compound increases the imino proton area of proton nuclear magnetic resonance (NMR) 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.
  • Other aspects of the invention provide a method for reducing cell apoptosis associated with BCL2 gene expression.
  • Such methods typically include contacting cells with a compound that increase i-motif structure of BCL2 gene to increase BCL2 gene expression thereby reducing cell apoptosis.
  • said compound reduces cell apoptosis by at least 25%o, typically at least 50%>, and often at least 75% compared to the amount of cell apoptosis in the absence of said compound.
  • said compound has a steroid core structure.
  • said compound has a steroid core structure.
  • 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
  • R 7 is (heterocyclyl)Ci_4 alkyl.
  • R 7 is (heterocyclyl)methyl.
  • 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.
  • Other aspects of the invention provide a method for enhancing therapeutic effectiveness of a cancer treatment in a cancer patient, said method comprising administering to the cancer patient undergoing a cancer treatment a compound that reduces i-motif structure of BCL2 gene.
  • said cancer treatment comprises chemotherapy, radiation therapy, or a combination thereof.
  • said i-motif structure occurs in the promoter region of BCL2 gene.
  • the present invention includes pharmaceutical 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.
  • 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.
  • 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.
  • 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).
  • hnRNP LL a transcriptional factor named hnRNP LL was shown to recognize the BCL2 i-motif and activated its transcription.
  • the binding of this protein to the i-motif provides the mechanism for the contrasting effects of IMC-48 and IMC-76. Therefore, taken together these observations make the dynamic equilibrium of the non-canonical DNA structure a target for small molecule control of gene expression.
  • 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 hnRNP 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.
  • 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
  • the hnRNP LL protein bound with high affinity to the BCL2 i-motif at pH 6.8 (Figure 9B).
  • hnRNP LL did not bind to duplex DNA formed with the cold complementary G-rich strand (Pu39WT) annealed to end-labeled Py39WT.
  • 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 ChlP 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 LL-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 hnRNP 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 RNA 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. Fluorescent secondary antibodies (IgG DyLight 800 or 680 conjugated from Thermo Scientific, Rockford, IL) were used and blots were visualized using developed with chemiluminescence.
  • BCL-2 Cell Signaling, Dancers, MA
  • ⁇ -actin Abeam, Cambridge, MA
  • MTS Cytotoxicity Assay The IC 50 values of the compounds for each cell line
  • 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 Purification of Recombinant 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. [0119] Streptavidin (Leinco Technologies, Inc., St. Louis, MO) (SA) was immobilized on a CM5 chip using standard amine coupling.
  • carboxy groups on the chip surfaces were activated with an injection of 0.05 M N-hydroxysuccinimide/0.2 M 1- ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride at a flow rate of 10 ⁇ / ⁇ for 7 min SA, diluted in 10 mM NaOAc (pH 5.5), 20 ⁇ g/mL, and pulsed over the surface at a flow rate of 10 ⁇ / ⁇ ⁇ until 1750 RU was achieved. Temperature was 25 °C and running buffer was 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% Tween 20.
  • 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 nM 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
  • 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 x 10 5 ) and BJAB cells (3 x 10 5 ) were treated with 2 ⁇ IMC-76 and IMC-48 for 24 h.
  • 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.

Abstract

The present invention provides a method for treating a clinical condition associated with underexpression of BCL2 gene. In particular, the present invention provides a method for treating a clinical condition associated with underexpression of BCL2 gene by administering a compound that increases i-motif structure of BCL2 gene thereby increasing the expression of BCL2 gene.

Description

METHOD FOR INCREASING BCL2 GENE EXPRESSION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S. Provisional Application
Nos. 61/939,809 and 61/939,813, both of which were filed February 14, 2014, and are incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under grant number
GM085585-01, CA153821 and T32CA09213 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
[0003] The present invention relates to a method for treating a clinical condition associated with underexpression of BCL2 gene. In particular, the present invention relates to treating a clinical condition associated with underexpression of BCL2 gene by administering a compound that increases i-motif structure of BCL2 gene.
BACKGROUND OF THE INVENTION
[0004] DNA secondary structures, such as 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. Specifically, G-quadruplexes are proposed to act as molecular switches that turn transcription on or off. Consistent with this role as a transcriptional regulatory element, G- quadruplexes within the c-MYC, c-KIT, KRAS, VEGF, PDGFA, PDGFR-β, HIF-la, c-MYB, and h TERT promoter regions have served as molecular targets for altering gene expression.
[0005] It is believed that the ability of 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.
[0006] Evasion of apoptosis, a hallmark of cancer, due to overexpression of BCL2
(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.
[0007] While there has been some research in the area of BCL2 gene modulation, a better understanding of how BCL2 gene expression is modulated would provide a new and useful therapeutic target. Accordingly, there is a continuing need for modulating BCL2 gene expression.
SUMMARY OF THE INVENTION
[0008] 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.
[0009] 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 method for therapeutic intervention based on shifting the equilibrium populations of BCL2 gene conformational isomers. In one particular embodiment, the method increases formation of the i-motif conformation of BCL2 gene compared to the flexible hairpin conformation of BCL2 gene. In some embodiments, the amount of relative increase in the i- motif (relative to the amount of the i-motif 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%.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] 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.
[0011] 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 DNATMC-76 complexes titrated with increasing amount of IMC-76, at 0, 2, 4, and 6 equivalents, respectively.
[0012] 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.
[0013] 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).
[0014] 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.
[0015] Figure 6 shows effect of IMC-76 and IMC-48 on BCL-2 protein levels in lymphoma cell lines. Western blot analysis of BCL-2 protein levels in BJAB, B95.8 and GRANTA-519 cell lines following 24 h treatment with IMC-76 (Panel A) or IMC-48 (Panel B). In both panels A and B, lanes 1 and 2, represent untreated and DMSO vehicle cell lysates, respectively. In panel A, lanes 3 and 4 contain lysates from cells treated with 0.25 and 0.5 μΜ IMC-76, respectively. In panel B, lanes 3 and 4 contain lysates from cells treated with 1 and 2 μΜ IMC-48, respectively. The western blots are representative of three independent experiments with β-actin as a loading control.
[0016] Figure 7 shows effect of IMC-76 on BCL2 mRNA levels in various breast carcinoma cell lines. The BCL2 mRNA levels as determined by qPCR for MCF-7, MCF- 7/TAMR and MDA-MB-231 breast carcinoma cell lines following 24 h treatment with IMC- 76. Percent change in mRNA levels were calculated relative to untreated controls. MCF-7 and MDA-MB-231 BCL-2 mRNA levels were normalized to β-actin and MCF-7/TAMR levels were normalized to GAPDH. *P < 0.02.
[0017] 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.
[0018] Figures 9A-9D show results confirming hnRNP LL as a BCL2 i-motif-binding protein. In particular, 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.
[0019] 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).
[0020] 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 μg 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.
[0021] 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.
[0022] 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. Upon addition of IMC-76, the flexible hairpin form is sequestered (A to B), resulting in depletion of the populations of the i-motif species. Conversely 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). Following this recognition event, there are hnRNP LL-driven changes in the interhelical conformations such that the two lateral loops are forced apart so that the further spaced apart RRMs 2 and 3 or 4 are able to bind to the 5 ' and 3 ' recognition sequences to form a stable complex (C to D). Last, 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. Alternatively 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).
[0023] 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.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Methods and Compounds of the Invention: Some aspects of the invention provide methods for treating a clinical condition associated with underexpression of BCL2 gene. Such methods include administering to a subject in need of such a treatment a compound that increases i-motif structure of BCL2 gene, thereby increasing the expression of BCL2 gene in the subject. Methods of the invention increases 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. By increasing the BCL2 gene expression in the subject, methods of the invention allow treatment of a clinical condition associated with
underexpression of BCL2 gene. "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. In some embodiments, said i-motif structure occurs in the promoter region of BCL2 gene. Exemplary clinical conditions that can be treated with methods of the invention include neurodegenerative diseases as well as other clinical conditions that are caused by underexpression of BCL2 gene. In particular, methods of the invention can be used to treat amyotrophic lateral sclerosis (ALS) or a neurodegenerative disease such as schizophrenia, Alzheimer's disease, Huntington disease and other neurodegenerative diseases caused by underexpression of BCL2 gene.
[0025] In some embodiments, the compound decreases 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 more often at least 90% when the ratio of said compound to BCL2 DNA is about 4: 1. Alternatively, the compound increases the imino proton area of proton nuclear magnetic resonance (NMR) 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.
[0026] Other aspects of the invention provide a method for reducing cell apoptosis associated with BCL2 gene expression. Such methods typically include contacting cells with a compound that increase i-motif structure of BCL2 gene to increase BCL2 gene expression thereby reducing cell apoptosis. Typically, said compound reduces cell apoptosis by at least 25%o, typically at least 50%>, and often at least 75% compared to the amount of cell apoptosis in the absence of said compound.
[0027] In some embodiments, said compound has a steroid core structure. Within these embodiments, in some in s of the formula:
Figure imgf000009_0001
I
where R1 is -ORa; R2 is H or a carbonyl moiety of the formula -C(=0)-Rb or -C(=0)-Rc- X1-C(=0)-Rb; R3 is hydrogen or -ORa; R4 is hydrogen or together with R3 forms =0; R5 is - ORa or -NHRd; R6 is -CH(NReRf)-Rg, -(C=0)-Rh-NReRf, -CH-(CH3)-R9 wherein R9 is branched or linear alkyl with 2 - 7 carbon atoms; or
Figure imgf000010_0001
wherein R is -H or linear or branched alkyl and n = 1 - 4; each Ra is independently H, alkyl or a carbonyl moiety of the formula -C(=0)-Rb; each Rb is independently H or alkyl; each of Rc and Rh is independently alkylene; Rd is H or -C(=0)-X2; each of Re and Rf is independently alkyl or optionally substituted aryl, heteroaryl or heterocyclyl, or Re and Rf together with the nitrogen atom to which they are attached to form an optionally substituted five- or six-membered heterocyclyl; Rg is alkyl; X1 is -O- or -N(Rb)-; and X2 is optionally substituted aryl, heteroaryl or heterocyclyl, or -NRbRb.
[0028] In some embodiments, R1 is -ORa, where Ra is H or -C(=0)CH3. Still in other embodiments, R2 is H, -C(=0)CH3, -COCH2OH, -COCH2OCOCH3, -COCH2OH. Yet in other embodiments, Rb is alkyl. In one particular embodiment, Rb is methyl. In other embodiments, R3 is H, -OH or together with R4 forms =0. In other embodiments, said compound is of the formula: R7-C(=0)-X3-R8 (Formula III), where R7 is optionally substituted (heterocyclyl)alkyl; R8 is optionally substituted cyclyl or aryl, or a steroidal moiety; and X3 is -O- or -NH-. In some embodiments, R7 is (heterocyclyl)Ci_4 alkyl.
Typically, R7 is (heterocyclyl)methyl. In some particular embodiments, heterocyclyl of R7 is piperidinyl or N-methylpiperidinyl. Often heterocyclyl of R7 is piperidin-l-yl or N- methylpiperidin-l-yl. Typically X3 is NH. Yet in other embodiments, R8 is C17-substituted steroid moiety or cyclohexyl, phenyl, or adamantyl, each of which is optionally substituted. In some particular embodiments, R8 is 17-(6-methylhept-2-yl)steroid moiety, cyclohexyl, phenyl, adamant- 1-yl, or 2,4,6-trimethylphenyl.
[0029] Still further, combinations of different embodiments of various groups described herein form other embodiments. In this manner, a variety of different
embodiments of compounds are embodied within the present invention.
[0030] "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. Exemplary 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. "Aralkyl" refers to a moiety of the formula -RbRc where Rb is an alkylene group and Rc 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. The terms "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. The terms "cycloalkylalkyl," "(cycloalkyl)alkyl," "cyclylalkyl" and "(cyclyl)alkyl" are used interchangeably herein and refer to a moiety of the formula -RdRe where Rd is an alkylene group and Re 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 terms "heterocyclyl" and "heterocycloalkyl" are used interchangeably herein and refer to a non-aromatic mono-, bi- or tricyclic moiety of three to fifteen ring atoms in which one or more, typically one, two or three ring atoms are heteroatoms selected from N, O, or S(0)n (where n is an integer from 0 to 2), the remaining ring atoms being C, where one or two C atoms can optionally be a carbonyl group (i..e, -C(=0)-). The heterocyclyl ring can be optionally substituted with one or more, typically one, two, or three, substituents. When two or more substituents are present in a heterocyclyl group, each substituent is independently selected. Exemplary 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.
[0031] When describing a chemical reaction, the terms "treating", "contacting" and
"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.
[0032] The term "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. Exemplary 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. The term "steroid core structure" refers to a moiety having the following general steroid ring system:
Figure imgf000012_0001
The steroid ring structure can be substituted and/or include one or more unsaturation.
[0033] In some embodiments, said clinical condition comprises cancer such as, but not limited to, melanoma, breast cancer, prostate cancer, chronic lymphocytic leukemia, and/or lung cancer.
[0034] Other aspects of the invention provide a method for enhancing therapeutic effectiveness of a cancer treatment in a cancer patient, said method comprising administering to the cancer patient undergoing a cancer treatment a compound that reduces i-motif structure of BCL2 gene. In some embodiments, said cancer treatment comprises chemotherapy, radiation therapy, or a combination thereof. In other embodiments, said i-motif structure occurs in the promoter region of BCL2 gene.
[0035] Administration and Pharmaceutical Composition: The present invention includes pharmaceutical 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.
[0036] In general, 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.
[0037] Typically, 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.
[0038] 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. The pharmaceutical 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.
[0039] 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. In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided 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. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suitable for oral administration.
[0040] Other forms suitable for oral administration include 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
preparations. 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.
[0041] 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. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles 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.
Alternatively, 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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
micronization. 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. The aerosol can conveniently also contain a surfactant such as lecithin. The dose of drug can be controlled by a metered valve. Alternatively 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 means of an inhaler.
[0047] When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient. For example, 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.
[0048] 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.
[0049] Other 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.
[0050] When it is possible that, for use in therapy, therapeutically effective amounts of a compound disclosed herein, as well as pharmaceutically acceptable salts thereof, can be administered as the raw chemical, it is possible to present the active ingredient as a pharmaceutical composition. Accordingly, the disclosure further provides pharmaceutical 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. When applied to a combination, 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. In accordance with another aspect of the disclosure there is also provided 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.
[0051] When the 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.
[0052] 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. Under negative supercoiling conditions, 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. They were found to differ either in the intercalation topology of the C-C+ base pairs or by the coexistence of a partially folded form with the i-motif. At neutral pH the partially folded form can coexist with the single-stranded form. For the purpose of this disclosure, 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. A recent Tm study on a wider range of presumed promoter i-motifs found this to be generally true, with exception of the c-kit C-rich sequence.
[0053] It is generally accepted that DNA predominantly exists in duplex form in cells.
However, under torsional stress imposed by active transcription, DNA can assume non- duplex structures. 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. The present inventors have discovered that some compounds (e.g., a pregnanol derivative and a class of piperidine derivatives) can modulate BCL2 gene expression by stabilizing either the i-motif or the flexible hairpin species. It was discovered that stabilization of the i-motif structure results in significant up-regulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. 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.
[0054] 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. These two compounds with a steroid core structure have opposite effects on BCL2 gene expression. The cholestane derivative, which stabilizes the flexible hairpin, results in repression of BCL2 expression in breast cancer and lymphoma cell lines. BCL2 repression leads to chemosensitization of lymphoma to etoposide and significant reduction in tumor growth in a Granta-519 lymphoma model in SCID mice.
[0055] 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.
[0056] 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. Both 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). In subsequent experiments, 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.
Figure imgf000020_0001
Table 1. Representative structures and FRET values for steroidal compounds.
Figure imgf000020_0002
Figure imgf000020_0003
* 10 equivalent compared to DNA
[0057] Structural analyses of these molecules suggest that substitution at C17 on ring
D is responsible for a similar effect. Compound 5776-0002, which possesses bulky substituents at Ri and R2 (at CI 7), showed the highest FRET value among the series. N056- 0003, a hydroxy analog (R3) of 5776-0002, showed a lower FRET value, indicating the stabilizing effect of the -OH group.
[0058] 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. Thus 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. [0059] To directly assess how IMC-76 and IMC-48 affect the i-motif structure, a ID
NMR of the imino region was examined following incremental addition of each of the compounds (Figures 3 A and 3B). Apparent destabilization of the BCL2 i-motif by IMC-76 was observed by NMR; even at pH 6.0, which favors the i-motif formation, addition of IMC- 76 clearly shifted the equilibrium to the hairpin species (Figure 2A). Without being bound by any theory, this species (called a flexible hairpin because direct unfolding of the i-motif from the central loop) appears to result in the formation of 5 Watson-Crick base pairs surrounding an open region, which is believed to be the binding site for IMC-76. NMR titration data showed that at 1 equivalent of IMC-76 the BCL2 i-motif signature peaks at -15.5 ppm decreased significantly; at 4 equivalents they almost completely disappeared (Figure 2A, traces 6 and 7). On the other hand, the flexible hairpin species increased upon IMC-76 addition (Figure 2A, traces 3-6) and is clearly observed at 3 °C (Figure 2A, trace 7). The flexible hairpin species (signature peaks at ~13 ppm) appeared to be more predominantly populated in the presence of IMC-76 at pH 6.0 (Figure 2A, trace 7) than in the absence of IMC-76 at the transition pH 6.6 (Figure 2A, trace 2). Thus IMC-76 appears to redistribute the population from the i-motif to the flexible hairpin by stabilizing the population that forms this species (Figure 2A, traces 3-6).
[0060] A comparison of NMR traces 2 and 7 in Figure 2A, determined at 3 °C, showed that binding of IMC-76 to the DNA produced very little disruption of the existing hairpin but further populated the flexible hairpin species at the expense of the i-motif, as does increasing the pH from 6.0 to 6.6. IMC-76 has a structural resemblance to a steroidal diamine, which has been shown to bind to unstacked base pairs and stabilize the poly (dA- dT) duplex. It is believed that IMC-76 binds in the non- Watson-Crick base-paired regions of the flexible hairpin adjacent to the GC bases because they have greater flexibility than the GC base-paired regions. 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.
[0061] In contrast, addition of IMC-48 causes a shift of the equilibrium from the flexible hairpin to the i-motif structure (Figure 2B). NMR titration data at 3 °C and 25 °C showed that, at the transition pH 6.6, with the incremental addition of IMC-48, the BCL2 i- motif signature peaks at -15.5 ppm clearly increased as compared to the flexible hairpin species whose imino proton peaks are between 12 and 13 ppm (Figure 2B, traces 2-6).
Again, the i-motif signals are sharper at the higher temperature of 25 °C (Figure 2B, left). Thus IMC-48 appears to redistribute the population from the flexible duplex to the i-motif by form stabilizing the i-motif population.
[0062] Equilibrium between the BCL2 i-Motif and the Flexible Hairpin Structure:
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. Conversely, the equilibrium is shifted from the flexible hairpin structure to the i-motif with the incremental addition of IMC-48 to the 2: 1 complex of IMC- 76 and BCL2 DNA (Figure 2D, traces 2-5); however, the shift in population to the i-motif species induced by IMC-48 (Figure 2D, trace 5) is not as significant as the shift in population to the flexible duplex species induced by IMC-76 (Figure 2C, trace 5). This result suggests that IMC-76 has a stronger effect in redistributing the populations of the two species toward the flexible duplex form. It is noted that the flexible duplex structure is clearly observed only at low temperature, i.e., 3 °C, due to the higher exchange rate with solvent of the imino protons in the flexible duplex form. Therefore, NMR data clearly demonstrate that IMC-76 and IMC-48 are antagonists to each other in shifting the equilibrium between the BCL2 i- motif and the flexible hairpin structures.
[0063] Inter action(s) That Lead to Stabilization of the Structure: Having established by ID NMR that IMC-76 interacts with the flexible hairpin and IMC-48 interacts with the i- motif, experiments were conducted to further chemically define the binding of these compounds to the two different species. CD analysis was initially carried out to determine the effect of IMC-48 and IMC-76 on the melting points of the BCL2 i-motif. Since the CD signatures for the i-motif and hairpin are very similar, only very small changes were seen upon addition of 1 and 2 equivalents of each compound. However, the changes were as anticipated: IMC-48 increased the melting point by about 1 °C, whereas IMC-76 decreased it by about 0.5 °C. In addition, IMC-76 and IMC-48 changed the molecular ellipticity in opposite directions; IMC-48 increased the molecular ellipticity, whereas IMC-76 decreased it. Overall, these results suggest that while 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.
[0064] 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.
-C(=0)-NH-R2
Figure imgf000023_0001
As can be seen, all four of these compounds produced substantially about the same reduction (10-15%) in FRET value as IMC-48 (15%). Whether one of these compounds (i.e., IMC-42) that reduced the FRET value can also lower BCL2 expression in MCF-7 cells was tested. The results showed that both IMC-48 and IMC-42 were of substantially equal potency in increasing BCL2 gene expression but the time course for lowering BCL2 was longer (48 h) for IMC-42 than IMC-48 (24 h). This is not unexpected since IMC-48 is a steroid and is likely to be actively taken up by the cells. [0065] Having established that the piperidine substituent at C3 is likely responsible for the stabilization of the BCL2 i-motif, experiments were carried out to determine where the binding site might be on this structure. First it seems likely that binding occurs within one of the lateral or central loops. The present inventors had previously shown that the thymines at positions 20, 21, and 24 in the central loop of the i-motif are involved in stacking interactions and are likely to be part of a capping structure that stabilizes the i-motif. In further support of the capping structure providing stability to the i-motif, mutations made to bases within the central loop (thymines T20, T21, and T24, and cytosines C22 and C23) produced a less stable i-motif structure (Δ Tm = -6 to -8 °C) as compared to that formed by the wild-type sequence. This led to consideration as to whether this loop might be the likely site for binding of IMC- 48. To test this possibility, two different mutant i-motifs were used in which either both lateral loops (Mut5 ',3 'L) or the central loop (MutCL) were mutated. CD measurements showed that while binding of IMC-48 and IMC-42 to the lateral loop mutant i-motifs still increased the molecular ellipticity and shifted the CD maxima to a higher wavelength similar to the wild-type sequence, there was no significant effect on the i-motif with mutations in the central loop. This data further implicated the central loop as the binding site for IMC-48 and IMC-42. To determine whether IMC-48 causes disruption of the central loop upon binding, the fluorescent nucleoside naphthodeoxyuridine (NdU) was used as a probe by substituting for thymines at positions 20, 21, 24, or 39. When IMC-48 was titrated into the i-motif, this led to about a 20% decrease in fluorescence of the NdU structure at T21 indicating that binding of IMC-48 binding caused a small but significant disruption of the central loop structure. These data indicated that the positively charged piperidine ring interacts with a phosphate on the backbone of i-motif DNA and the small ring structure binds in the vicinity of T21 in the central loop to further stabilize the structure and increase the population of this species.
[0066] IMC-48 and IMC-76 Produced Opposite Effects on BCL2 Gene Expression:
The biological effect of the 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 IC50 values to avoid cytotoxicity (Figure 5 and Table 2). IMC-76 significantly reduced BCL2 mRNA levels at 0.25 and 0.5 μΜ in the B95.8 cells by 56% (P = 0.04) and 54% (P = 0.001) (Figure 3B, upper) and to a lesser extent in the GRANTA-519 cells by 23% (P = 0.02) and 33% (P = 0.02) (Figure 3B, middle). 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). In contrast, treatment with IMC-48, the i-motif- interactive compound, resulted in the upregulation of BCL2 gene expression in BJAB cells, with a 220%) (P = 0.04) increase at 2 μΜ, but no significant dose-dependent effect was observed in GRANTA-519 and B95.8 cells (Figure 3C, lower, middle, and upper,
respectively). A moderate increase in BCL2 mRNA (65%>, P = 0.02) was observed for GRANTA-519 only at the 1 μΜ dose (Figure 3C, middle). This BCL2 activation in BJAB cells by IMC-48 also occurred at the protein level (Figure 6). Results similar to those found in the lymphoma cell lines were found for IMC-76 in breast cancer cell lines that either overexpressed BCL2 or had basal levels of BCL2 (Figure 7; Table 2).
Table 2. IMC-76 and IMC-48 IC50 values at 96 h for breast carcinoma and lymphoma cell
Figure imgf000025_0001
ND: not determined
[0067] Chemosensitization: Repression of BCL2 through transcriptional modulation by IMC-76 should also result in chemosensitization of the etoposide-resistant lymphoma cell line B95.8. The B95.8 cells exhibited resistance to apoptosis following treatment with etoposide alone; however, upon co-treatment with IMC-76 (0.25 μΜ), caspase-3 activity was significantly increased (2.5-fold, P = 0.03) in B95.8 cells (Figure 3D, upper). A BH-3 mimetic from Abbott Laboratories, ABT-737 (0.25 μΜ), was used in parallel as a positive control and demonstrated similar chemosensitizing effects (2.2-fold increase, P = 0.03) in the
B95.8 cell line (Figure 3D, upper). Similar effects were observed with combination treatment of etoposide with IMC-76 (2.5 fold increase, P = 0.04) or ABT-737 (2.2 fold increase, P =
0.04) in the GRANTA-519 cell line (Figure 3D, middle). There was no significant effect on apoptotic induction by etoposide on the sensitive BJAB parental cell line with IMC-76 or
ABT-737 treatment (Figure 3D, lower). It is also important to note that IMC-76 and ABT- 737 single treatment, as well as the DMSO vehicle control, did not induce apoptosis relative to untreated controls.
[0068] This chemosensitization effect of IMC-76 was also observed using another standard chemotherapy agent, cyclophosphamide (Cytoxan) in the GRANTA-519-resistant lymphoma cell line. Similar to the chemosensitization to etoposide, IMC-76, when combined with cyclophosphamide, induced a significant increase in caspase-3 activity (1.9-fold increase, P = 0.01) compared to cyclophosphamide alone (Figure 3E). Confirmation of the antagonistic effect on BCL2 expression at the gene and protein levels (as well as occurrence of the shift in DNA secondary structure species equilibrium in the presence of both IMC-76 and IMC-48) were observed when the cells were treated with IMC-48 alone and with IMC-76 in the presence of Cytoxan. As expected, IMC-48 treatment with Cytoxan did not
significantly (P = 0.72) induce caspase-3 activity since this compound stabilizes the i-motif leading to an increase in BCL2 expression (Figure 3E). The differential effect of these two opposing compounds on increasing Cytoxan-induced apoptosis was significant (P = 0.04). When the cells were treated with both compounds concurrently with Cytoxan at a 1 : 1 ratio, the apoptosis-inducing effect of IMC-76 was diminished by the presence of IMC-48 (P = 0.04) and the caspase-3 activity resembled that of basal levels (Figure 3E). This further demonstrates the antagonistic nature of these two compounds in interacting with the BCL2 i- motif.
[0069] Induction of Chemosensitivity: In a maximum tolerated dose study, the in vivo efficacy of IMC-48 to downregulate BCL2 expression was evaluated in SCID (severe combined immunodeficient) GRANTA-519 lymphoma xenograft mice that were treated at fractionated doses of 6 mg/kg, 8 mg/kg, and 10 mg/kg for five days (N = 3). When averaged, the two responsive mice from the 6 and 8 mg/kg treatment group displayed a 20% decrease in BCL2 mRNA levels while a greater decrease of 48% was found in the 10 mg/kg mice (Figure 4A, left). 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 significant 65% growth inhibition (P = 0.0001) was observed for lymphoid tumors that received concurrent treatment of IMC-48 and etoposide 12 days post last drug administration, while neither monotherapy of etoposide (30%) or IMC-48 (14%) resulted in a significant reduction in tumor growth. There was no significant weight loss in mice treated with IMC-48 alone or in combination with etoposide (Figure 4B, right).
[0070] Conclusion: The intrinsic dynamic state of the i-motif and its associated equilibrating flexible hairpin is similar in many respects to the dynamic nature of R A. Compounds that bind preferentially to one or another of these equilibrium forms cause a repopulation of the two species. Furthermore, in vitro experimental results with IMC-48 and IMC-76 indicate that this redistribution of two equilibrating DNA species leads to contrasting transcriptional consequences on BCL2 expression. This discovery shows involvement of proteins, which would also recognize and bind selectively to the i-motif and the flexible hairpin to control gene transcription, similar to the observed effects of IMC-48 and IMC-76. In another experiment, a transcriptional factor named hnRNP LL was shown to recognize the BCL2 i-motif and activated its transcription. The binding of this protein to the i-motif provides the mechanism for the contrasting effects of IMC-48 and IMC-76. Therefore, taken together these observations make the dynamic equilibrium of the non-canonical DNA structure a target for small molecule control of gene expression.
[0071] Study o f BCL2 i-Moti f and hnRNP LL: The C-rich strand of the cis-regulatory element in the BCL2 promoter element is highly dynamic in nature and can form either an i- motif or a flexible hairpin. Under physiological conditions these two secondary DNA structures are in an equilibrium mixture, which can be shifted by the addition of a compound that trap out either the i-motif (e.g., IMC-48) or the flexible hairpin (e.g., IMC-76). In cellular experiments the addition of these molecules had opposite effects on BCL2 gene expression and furthermore these compounds are antagonistic. Disclosed herein also is a transcriptional factor that recognizes and binds to the BCL2 i-motif to activate transcription. In particular, 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. In subsequent experiments, it was discovered that IMC-48 and IMC-76 have opposite, antagonistic effects on the formation of the hnRNP LL-i-motif complex as well as on the transcription factor occupancy at the BCL2 promoter. Thus, it is believed that 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.
[0072] While there is a relatively an extensive knowledge in G-quadruplexes in telomeric sequences, promoter elements, and 5 'UTRs, similar research is lacking for the complementary DNA secondary structure. In promoter elements where duplex DNA is found, the possibility exists that the G-quadruplex and the i-motif form on opposite strands, but whether they can co-exist or are mutually exclusive remains unresolved, except in the case of the insulin promoter where the formation of the two structures is mutually exclusive. If the latter were the case more generally, then one might imagine that the G-quadruplex could act as a signal to silence gene expression, as is the case with the MYC promoter, and the i-motif as an activator signal. In support of this, the activating transcriptional factor hnRNP K binds to the CT boxes on the C-rich strand in the MYC promoter and induces MYC expression.
[0073] As discussed, DNA secondary structures serve as switches to turn gene transcription on or off. In fact, 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) that bound to the i-motif species to populate this species relative to the flexible hairpin increased BCL2 gene expression. In contrast, the compounds (such as IMC-76) that selectively bound to the flexible hairpin species decreased gene expression. 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.
[0074] The present inventors have discovered that 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. Importantly this principle was shown at both the level of the DNA species bound to hnRNP LL in solution as well as at the cellular level. These results show that the BCL2 i- motif can be considered as a molecular switch similar in principle to a riboswitch found in RNA.
[0075] Results and Discussion: Directly upstream (-25 bases) from the BCL2 PI promoter is a GC-rich element known to form G-quadruplex and i-motif structures. Under negative superhelicity induced by transcriptional activity it can be expected that either the i- motif or the G-quadruplex will exist in the promoter element. Previous in vitro studies using synthetic oligomers demonstrate that the BCL2 G-rich promoter element forms three different G-quadruplexes; the major one exhibits a mixed parallel/antiparallel structure. The opposite strand is highly dynamic existing as a mixed population of two molecules at a pH of 6.8, an i- motif and a flexible hairpin. The relationship between these two DNA secondary structures, the interaction of IMC-48 and IMC-76, and the subsequent effect on BCL2 gene expression are illustrated in Figure 8.
[0076] Specific proteins such as nucleolin and NM23-H2 recognize and bind to G- quadruplexes in promoter elements. 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.
[0077] 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.
Ninety- five proteins were identified that bound either to the wild-type BCL2 i-motif-forming sequence (35 proteins, Table 3), a mutant (20 proteins, Table 4) or both sequences (40 proteins, Table 5). 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. While hnRNP LL has not been extensively studied, related protein 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.
Table 3. Proteins purified using Py39WT BCL2 i-motif biotinylated oligomer-streptavidin bead com lex and identified b LC/MS/MS.
Figure imgf000030_0001
Figure imgf000031_0001
Table 4. Proteins purified using a Py39MutT BCL2 i-motif biotinylated oligomer- stre tavidin bead com lex and identified b LC/MS/MS.
Figure imgf000031_0002
Table 5. Proteins purified that bound both the Py39WT and Py39MutT BCL2 i-motif biotin lated oli omer-stre tavidin bead com lex and identified b LC/MS/MS.
Figure imgf000031_0003
FUS Isoform short of RNA-binding protein FUS IPI00221354
NM23-H1 Isoform 1 of nucleoside diphosphate kinase A IPI00012048
HMG-B2 High-mobility group protein B2 IPI00219097
HMG-B3 High-mobility group protein B3 IPI00217477
ILF-2 Interleukin enhancer-binding factor 2 IPI00005198
ILF-3 Isoform 5 interleukin enhancer-binding factor 3 IPI00219330
ELAV-like cDNA FLJ60076, highly similar to ELAV-like protein 1 IPI00301936
Translation or Protein Folding-Related
eEFl-A Elongation factor 1-a IPI00025447
DDBP1 DNA damage-binding protein 1 IPI00293464
DDBP2 Isoform 1 DNA damage-binding protein 2 IPI00021518
HSP70-1 Heat shock 70 kDa protein 1 IPI00304925
HSC71 Isoform 1 of heat shock cognate 71 kDa protein IPI00003865
PPIase A Peptidyl-prolyl cis-trans isomerase A IPI00419585
PPIase B Peptidyl-prolyl cis-trans isomerase B IPI00646304
PDIA3 Protein disulfide-isomerase A3 IPI00025252
PDI Protein disulfide-isomerase IPI00010796
PA2G4 Proliferation-associated protein 2G4 IPI00299000
SDN1 Staphylococcal nuclease domain-containing protein 1 IPI00140420
Energy and Purine Metabolism
PGK1 Phosphoglycerate kinase 1 IPI00169383
ACLY ATP-citrate synthase IPI00021290
GPI Glucose-6-phosphate isomerase IPI00027497
PURH Bifunctional purine biosynthesis protein PURH IPI00289499
Cell Surface Adhesion, Migration, or Organization-Related
RCC2 Regulator of chromosome condensation 2 IPI00465044
CK6B Keratin, type II cytoskeletal 6B IPI00293665
CK1 Keratin, type II cytoskeletal 1 IPI00220327
CK10 Keratin, type I cytoskeletal 10 IPI00009865
CK9 Keratin, type I cytoskeletal 9 IPI00019359
CK2 Keratin, type II cytoskeletal 2 epidermal IPI00021304
CLF-1 Cofilin-1 IPI00012011
ACTA1 Actin, cytoplasmic 1 IPI00021439
FSCN1 Fascin IPI00163187
Isoform 2 of 4F2 cell-surface antigen heavy chain IPI00027493
32 kDa protein IPI00176692
RKIP Phosphatidylethanolamine-binding protein 1 IPI00219446
[0078] To investigate hnRNP LL for its possible involvement in BCL2 transcriptional modulation, the effects of siRNA knockdown in MCF-7 breast cancer cells were studied. Relative mRNA levels of hnRNP LL and BCL2 were determined by qPCR after treatment with hnRNP LL siRNA. Significantly decreased BCL2 mRNA levels resulted from hnRNP LL knockdown (Figure 9A). [0079] Bindins of hnRNP LL to the BCL2 i-Motif. Mobility shift assays (Figures 13B and C) were used to determine whether hnRNP LL bound to the BCL2 i-motif specifically. The hnRNP LL protein bound with high affinity to the BCL2 i-motif at pH 6.8 (Figure 9B). The cold BCL2 i-motif (Py39WT) competed with the 32P-labeled BCL2 i-motif; as expected the cold mutant i-motif oligomer (Py39MutT) did not compete for hnRNP LL binding (Figure 9C). As well, hnRNP LL did not bind to duplex DNA formed with the cold complementary G-rich strand (Pu39WT) annealed to end-labeled Py39WT.
[0080] Using SPR analysis (Figures 9D and 14), hnRNP LL bound strongly to
Py39WT with a KD value of 19.4 pM at pH 6.5. This disassociation was increased by -3.6- fold to 69.8 pM at pH 7.9. For the Py39MutT, the KD value was 2.5-fold higher at each pH compared to the wild-type.
[0081] 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. Of these four mutated i-motif sequences, the one having both lateral loops mutated (Mut5 ',3 'L; 35%) was the least effective competitor, while the one having the central loop mutated (MutCL; 73%) was the most effective. While the two individually mutated loop oligomers (Mut5 X and Mut3 X) were of intermediate potency in competing with the wild-type for hnRNP LL binding to the BCL2 i-motif, Mut5 X (48%) appeared less effective than Mut3 X (64%). To complement these experiments, the biological significance of the lateral loops were also determined by investigating whether hnRNP LL knockdown depended on the wild-type loop sequences. Two mutant luciferase constructs were prepared, one in which both lateral loops were mutated (Mut5 ',3 X) and a second in which the central loop was mutated (MutCL). While knockdown of the hnRNP LL still had an inhibitory effect on luciferase activity with the wild-type and MutCL promoter constructs, there was no significant effect on reporter activity with the Mut5 ',3 X (Figure 10). The siRNA knockdown of reporter activity (~25%>) could be considered modest; however, this is probably due to the limited (50-60%>) knockdown of hnRNP LL and suggests that activation of BCL2 expression may involve other transcriptional factors. To determine the relative importance of the sequence of each of the lateral loops, a similar EMSA competition experiment was carried out in which either the 5 ' or 3 ' sequence was swapped out (Mut6) or both lateral loops carried either the 5 ' (Mut6-1) or 3 ' (Mut6-2) loop sequences. The histograms of band quantification did not reveal significant differences for any of the sequences (64% for Mut6, 65% for Mut6-1, and 70% for Mut6-2), indicating that the variance observed between Mut6-1 and Mut6-2 may be related to the 5 ' or 3 ' positions of the lateral loop and the associated R Ms rather than sequences.
[0082] Binding ofhnRNP LL to the BCL2 i-Motif. There was a significant decrease in the ellipticity of Py39WT at 286 nm in the presence of hnRNP LL in a concentration- dependent manner and a shift to a lower wavelength by about 2.2 nm at 2 equivalents as detected by CD analysis. This suggests a partial unfolding of the i-motif or conversion to another topological form by hnRNP LL. To a lesser extent, the CD signal of Py39MutT was also decreased by the binding of hnRNP LL and shifted to a higher wavelength by about 1 to 2 nm at 2 equivalents. To determine the optimum distance between the two lateral-loop binding sites for competition with Py39WT, a series of oligomers containing the two consensus sequence binding sites connected by variable (2-17 nucleotides) spacers was used. A 13 -nucleotide spacer was found to be optimal for competition with the end-labeled
Py39WT, which is the exact nucleotide distance between the two lateral loops in the wild- type sequence. It is important to note that the molar ratio of the unlabeled, unstructured 39- mers to the labeled 39-mer i-motif was 150: 1. Pre -organization of the consensus binding sequences into the lateral loops of the folded i-motif provides a significant entropic and kinetic binding advantage. Based on these finding, it appears that a key function of the i- motif folded structure is to provide a rigid chemical scaffold upon which to display the pre- organized lateral loops for optimum kinetic advantage for binding of hnRNP LL. In addition the sequential recognition and binding of not just one but both lateral loops also provide a significant kinetic advantage. On the basis of these EMSA experiments it is believed that the RRMs of 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.
[0083] The structure of the Py39WT oligomer after hnRNP LL binding was examined by bromine footprinting (Figure 11). Binding of hnRNP LL to the BCL2 i-motif changed the cleavage pattern of Py39WT following bromination/piperidine treatment (compare lanes 2 and 4 in Figure 11). Cytosines in run I were more cleaved while other cytosines in runs III to IV were less cleaved with increasing concentrations of hnRNP LL. Bromine footprinting of the same sequence in the presence of IMC-76 indicated the opposite effect on runs III and IV. This suggests that the unfolded form induced by hnRNP LL is not the partial hairpin. Since runs III to IV are positioned between the 5 ' and 3 ' lateral loops, which the spacer experiment demonstrated must be unfolded in the hnRNP LL-bound species, bromine footprinting inhibition suggests that they are more protected by close association with hnRNP LL than even the lateral loops (Figure 11).
[0084] The i-motif unfolding activity of hnRNP LL was further confirmed by a quencher-based FRET assay. In this experiment 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. In addition, 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.
[0085] The binding of hnRNP LL 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. Significantly, 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. Thus, 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.
[0086] Mutually Exclusive Binding to the i-Moti f: The partitioning of biological molecules between two equilibrating species in which only one is biologically active is well known in the RNA world. These can act as switches if the chemical equilibrium can be changed by the preferential sequestration of one of the forms by a small molecule. To determine whether a similar mechanism might operate in a system consisting of two equilibrating DNA species, the biological outcome (transcriptional silencing or activation) was explored using a small molecule that bound preferentially to each of the DNA forms. Characterization of the ternary interactions between the DNA, protein, and each small molecule in a cell-free system permitted extension into a cellular system.
[0087] 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. In cells, IMC-76 decreases the i-motif population in the promoter element and thus reduces hnRNP LL promoter occupancy. In contrast, 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. Experiments were conducted using EMSA, knockdown of hnRNP LL, and ChlP analysis was also performed.
[0088] The results from the EMSA experiment in which different amounts of IMC-76 were incubated at pH 6.5 with the BCL2 i-motif and its equilibrating species for 3 h prior to the addition of hnRNP LL are shown in Panel A in Figure 12. In the absence of IMC-76 and hnRNP LL, there are two conformationally different oligomer species separated in the gel. It is believed that species 1 , the predominant form, is most likely the i-motif, which leaves species 2 as the flexible hairpin. Upon addition of hnRNP LL, species 1 was depleted preferentially to form the hnRNP LL high-mobility-shifted complex. However, as IMC-76 concentration was increased, the amount of the hnRNP LL-BCL2 i-motif complex decreased, and species 2, putatively the flexible hairpin, increased at the expense of species 1, the presumed i-motif. This is in accord with the idea that IMC-76 and hnRNP LL compete for the pool of equilibrating species to trap (IMC-76) or remodel (hnRNP LL) the i-motif to an unfolded species. In a parallel experiment the effect of IMC-48 on the distribution of the three species was determined and shown in Panel B of Figure 12. 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 LL-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.
[0089] To investigate the IMC-76 and IMC-48 cellular effects, which are known to affect BCL2 transcription, siRNA and ChlP experiments were performed. First, the potentially additive or subtractive inhibitory effects of hnRNP LL siRNA together with IMC- 76 or IMC-48 were determined on BCL2 mRNA transcription, following knockdown of hnRNP LL. Second, to determine the effects of IMC-76 or IMC-48 on promoter occupancy by Spl and hnRNP LL, ChlP analysis was performed using MCF-7 cells. While the treatment of 50 nM hnRNP LL siRNA alone significantly decreased BCL2 mRNA levels by 24%, addition of either 0.5 or 2 μΜ of IMC-76 further decreased the mRNA levels to a total of 33% and 47%, respectively. In contrast, IMC-48 reversed the inhibitory effects of the hnRNP LL siRNA. This is expected because both knockdown of hnRNP LL and depletion of the i- motif population by IMC-76 should be additive in lowering transcription, although they act on different targets. In contrast, the effect of IMC-48 in cells should antagonize the inhibitory effects of BCL2 mRNA expression knockdown.
[0090] To directly assess the effect of IMC-76 and IMC-48 on recruitment of transcriptional factors to the BCL2 promoter, which is proposed to contain the i-motif- forming element, a ChIP assay was performed on Spl and hnRNP LL using MCF-7 cells and BJAB cells, respectively. The MCF-7 cells, which overexpress BCL2, were used to determine the inhibitory effect of IMC-76 on BCL2 transcription. Alternatively, the BJAB cells, which only express basal levels of BCL2, were used to evaluate the activating effect of IMC-48 on the BCL2 transcription. Spl is a ubiquitous transcription factor bound to the GC-rich region in gene promoters. MCF-7 and BJAB cells were treated with IMC-76 and IMC-48 (at 0.5 and 2 μΜ) 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. In contrast, IMC-48 increased the promoter occupancy of both Spl and hnRNP LL in BJAB cells. To ensure that the effect of IMC-76 on promoter occupancy by Spl and hnRNP LL was not due to inhibition of transcription of these proteins, qPCR was carried out. In a similar way, the effect of IMC-48 on the transcription level of Spl and hnRNP LL was tested with BJAB cells. In addition, immunoprecipitation (IP) experiments were carried out for both Spl and hnRNP LL to verify antibody specificity.
[0091] 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. To extend these studies to examine what happens at the promoter level to the transcriptional factors that bind to the BCL2 regulatory element 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 results showed that the decreased promoter occupancy by both Spl and hnR P LL in MCF-7 cells produced by IMC-76 was reversed by IMC-48 in a concentration-dependent manner, illustrating an antagonistic relationship between IMC-76 and IMC-48 at the promoter level. This result, together with the results from experiments carried out at the solution level using NMR and at the cellular level using chemosensitization to cyclophosphamide, provide very strong evidence for direct competition between IMC-76 and IMC-48 for the two equilibrating populations of the BCL2 i-motif and flexible hairpin resulting in the cellular consequences mediated via hnRNP LL.
[0092] Molecular Switch Model for the BCL2 i-Motif. Of the non-canonical DNA structures, 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 ρΚΆ 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
parameters. What is critical for the proposed role of i-motifs as molecular switches in transcriptional regulation is that their dynamic nature is such that they can easily move between folded (i-motif) and unfolded (hairpin) populations of molecules under physiological conditions. Thus factors such as those described above that influence the topology of the deoxyribose backbone of the intercalated C-CH+ base pairs likely play critical roles in determining the dynamic nature of promoter i-motifs.
[0093] In cellular systems, where both transcriptionally induced negative
superhelicity and molecular crowding can occur, this molecular plasticity of i-motifs in promoter elements is likely to be evident under physiological conditions, even without considering the presence of transcriptional factors such as hnRNP LL, which can recognize and then unfold the i-motif to form a thermodynamically stable species. Thus, it is believed that promoter i-motifs have dynamic properties more like RNA secondary structures than what are typically associated with DNA. Both small molecules (e.g., IMC-76 and IMC-48) and a transcriptional factor (hnRNP LL) can either change the relative population states of the i-motif and its equilibrating conformers or, in the case of hnRNP LL, also drive changes in the interhelical conformation of the i-motif to bind most stably to an alternative conformation not originally present. 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.
[0094] In the dynamic transitional system shown (Figure 13 A), it is believed that two predominant conformational states exist, where the flexible hairpin can coexist with either a single-stranded form or the fully folded species (i-motif). For i-motif-forming sequences at neutral pH, both the partially folded and single-stranded states coexist; and under molecular crowding conditions the i-motif conformation can exist even at neutral conditions. Under negative superhelicity, the i-motif has been observed in the MYC promoter under
physiological conditions. For the BCL2 promoter sequence, at pH 6.6 the flexible hairpin and i-motif forms can be observed both by 1H NMR and in an EMSA gel. Incremental IMC-76 addition sequesters the flexible hairpin form, which contains five GC base pairs. By analogy with the binding of a steroidal diamine to a poly(dA-dT) duplex, IMC-76 most likely binds in the non- Watson-Crick base pair regions where unstacked base pairs exist capped at either side by GC base pairs (Figure 13B). IMC-76 sequestration of the flexible hairpin species will deplete the i-motif. This redistribution of the conformation species results in a reduction in the amount of the hnRNP LL-shifted band, which in a cellular context would result in reduction of BCL2 transcriptional activation and chemosensitization. IMC-48 does the opposite and increases the amount of hnRNP LL shifted band. It does this by binding to the central loop of the BCL2 i-motif and further constrains the lateral loops through which hnRNP LL recognizes and binds before unfolding the structure. This would further accelerate the kinetic step and lead to enhanced transcriptional activation of BCL2. Furthermore, it is likely that under varying extents of negative superhelicity produced during transcriptional firing the intrinsic dynamic behavior of the i-motif and its equilibrating conformational forms will be even more accessible. This may be important when larger energetic barriers are present, such as in the disruption of the cytosine-cytosine base pairs. The i-motif folding and unfolding kinetics, the latter requiring disruption of base pairing, is slow in comparison to RNA elements like riboswitches. However, in cellular promoter elements, where i-motif- binding proteins are present together with dynamic forces that result from negative superhelicity, the kinetics may be much faster.
[0095] 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 (1/2 or 2/3) is required for the high-affinity binding of hnRNP L to RNA. The competition experiments demonstrate that pre-organization of the binding sequences in the i-motif lateral loops confer entropic and kinetic advantages for hnRNP LL binding. Additionally, while the RRM2 of hnRNP L is sufficient to achieve RNA binding specificity, an additional RRM is required for complex stabilization.
[0096] A comparison of the role of hnRNP L in the RNA switch that regulates VEGF expression with the role of hnRNP LL in the regulation of BCL2 expression provides two insightful analogies. First, the 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. Second, 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.
[0097] 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). 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.
[0098] 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. First, there is a CA element in the upstream region that is a potential hnRNP L binding site; the binding of the more ubiquitous hnRNP L to this element may result in a looping structure with formation of a heterodimer with hnRNP LL to activate transcription. Second, 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.
[0099] Conclusion: The intrinsic dynamic state of the i-motif, similar in many respects to the dynamic nature of RNA, makes the dynamic equilibrium of the non-canonical DNA structure an attractive target for small molecule control of gene expression. It is important to note that the biological effects observed were consistent with i-motifs in cells. For the BCL2 i-motif, the mutual exclusivity of IMC-76 and hnRNP LL for targeting different conformational forms of the equilibrating i-motif allows the repression of BCL2 gene expression and chemosensitization of drug-resistant lymphoma and breast cancer cells using a steroid molecule. Conversely enhanced expression of 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.
[0100] Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting. In the Examples, procedures that are constructively reduced to practice are described in the present tense, and procedures that have been carried out in the laboratory are set forth in the past tense.
EXAMPLES
[0101] Compounds: The NCI Diversity Set of compounds was obtained from the
National Cancer Institute, National Health Developmental Therapeutics Program (Bethesda, MD). 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.
[0102] 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 μΜ) was incubated with compounds from the NCI diversity set (5 μΜ) 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. Interactive compounds were validated by FRET analyses performed with BCL2 i-motif mutant, c-MYC i-motif, VEGF i-motif, and BCL2 duplex, and G-quadruplex probes were placed in buffer at pH 6.3 with or without KC1 at a concentration of 1 μΜ and incubated at room temperature (25 °C) for one hour to allow for i-motif or G- quadruplex formation. The BCL2 duplex was formed by annealing the BCL2 i-motif and G- quadruplex sequences in a 50 mM Na cacodylate buffer at pH 7.1 without KC1. For IMC-48 mimic compounds (Chembridge), the BCL-2 i-motif probe (1 μΜ) at 50 mM Na cacodylate (pH 6.3) was heated for 5 min at 95 °C and cooled on ice.
[0103] Circular Dichroism: CD analyses were conducted on a Jasco-810
spectropolarimeter (Jasco, Easton, MD) using a quartz cell of 1 mm optical path length. 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 μΜ 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 λ of the maximum molar ellipticity). CD spectra were baseline corrected by subtracting a buffer alone or a buffer with compound.
[0104] NMR Studies: The DNA oligonucleotide was purchased from Euro fins MWG
Operon. The final NMR samples were prepared in 10%/90% D2O/H2O 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 d6-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.
[0105] 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 μΜ 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.
[0106] Bromine Footprinting: Briefly, BCL2 i-motif wild-type oligonucleotide was
5 '-end-labeled with [γ-32Ρ] ATP, purified, and incubated with or without IMC-76 (15 μΜ) in a 50 mM Na cacodylate buffer (pH 6.1). The samples were incubated with molecular E¾ formed in situ by mixing an equal molar concentration (50 mM) of KBr with KHSO5 for 20 min and terminated by the addition of 60 of a 0.6 M sodium acetate and calf thymus DNA (10 mg/mL) solution. Any unreacted E¾ was removed in subsequent ethanol precipitation steps. After ethanol precipitation, the DNA pellet was dried and resuspended in 30 μΐ, of a 100 mM piperidine. Samples were heated at 90 °C for 20 min to induce bromination-specific strand cleavage, dried, and resuspended with alkaline sequencing gel loading dye. The bromination-specific strand cleavage was visualized on a sequencing gel (20% PAGE with 7 M urea). A pyrimidine-specific reaction was performed using hydrazine to generate a cytosine-sequencing marker.
[0107] Cell Culture: MCF-7, MDA-MB-231, and GRANTA-519 cell lines were purchased from the American Type Culture Collection (Manassas, VA). The 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.
[0108] Real-Time RT-PCR: Cells were harvested following 24 h treatment with IMC-
76 (0.125, 0.25, 0.5 μΜ), IMC-48 (0.25, 0.5, 1, 2 μΜ), and IMC-42 (0.5, 2 μΜ). Cells untreated and treated with DMSO vehicle control were used to determine basal BCL2 expression levels. Total RNA was isolated with a Qiagen RNeasy Kit (Valencia, CA) according to the manufacturer's protocol. Reverse transcription was performed using the Quantitect reverse transcription kit (Qiagen) or PrimeScript RT Kit with gDNA Eraser (Takara) as per the manufacturer's protocol. Real-time PCR was conducted using MyCycler (Bio-rad). Ct values were normalized to either β-actin or GAPDH and compared to the untreated controls. TaqMan probes were used for BCL2 (HsOO 153350 or Hs00608023) and GAPDH (Hs02758991) PCR amplification. Samples analyzed with β-actin as a reference gene. For IMC-42 (0.5, 2 μΜ), real-time PCR was performed using Rotor-Gene Q (Qiagen).
[0109] Western Blot Analysis: Expression of BCL2 protein was examined in cell lines at basal levels or following 24 h treatment with IMC-76 or IMC-48. Untreated and DMSO- treated cells were used as controls. Protein lysates were obtained by incubation with RIP A buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP40, 0.25% Na deoxycholate, 1 mM PMSF, 1 x Roche complete mini protease inhibitor cocktail) followed by sonication and centrifugation at 14,000 rpm for 10 min at 4 °C. Protein concentrations were determined by the BSA assay (Pierce, Rockford, IL). Protein bands were resolved on a precast 10%> sodium dodecyl sulfate polyacrylamide gel (BioRad, Hercules, CA) from 40 μg 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 β-actin (Abeam, Cambridge, MA) which were used at a dilution of 1 : 1000 and 1 :5000, respectively. Fluorescent secondary antibodies (IgG DyLight 800 or 680 conjugated from Thermo Scientific, Rockford, IL) were used and blots were visualized using developed with chemiluminescence.
[0110] MTS Cytotoxicity Assay: The IC50 values of the compounds for each cell line
(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
(Promega, Madison, WI).
[0111] Caspase-3 Activity Assay: Caspase-3 activity was evaluated using the
ApoAlert Caspase-3 Plate Assay as per the manufacturer's specifications (Clontech,
Mountain View, CA).
[0112] In Vivo Xenograft Studies: Every month mice were screened by ELISA serology for mycoplasma, mouse hepatitis virus, pinworms, and Sendai virus. Cells were tested for mycoplasma and viability (>95%) prior to injection (10 x 106 cells/ 100 sterile saline). SCID mice for the MTD study (four groups of N = 3) and the combination study (four groups of N = 12) were injected with GRANTA-519 mantle lymphoma cells subcutaneously in the left flank. IMC-76 and/or etoposide were administered by intraperitoneal injection every day for a total of five days. As tumors developed, SC tumors were measured for tumor volume estimation (cm3 or mm3) in accordance with the formula a2 x b/2, where a equals the smallest diameter and b is the largest diameter. Tumors were only allowed to reach 2000 mm3. 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.
[0113] Statistical Analysis: Statistical significance (P <0.05) was evaluated using a two-tailed Student's t-test except for the in vivo study. Data are presented as mean ± standard error from three independent experiments.
[0114] 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 μg 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 MgCl2, 1 mM EDTA, 10% glycerol) plus l protease inhibitor cocktail overnight, rotating. Following overnight incubation, the beads were washed in Buffer B. The mutant oligomer-conjugated beads were incubated with 500 μg 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/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, Tucson, 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.
[0115] Purification of Recombinant 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-gami™ B (DE3) pLysS cells (Novagen). The expression of hnRNP LL was induced by 0.1 mM IPTG
(isopropyl β-D-l-thiogalactopyranoside) overnight at room temperature. Harvested cells were resuspended in a lysis buffer (50 mM NaH2P04 (pH 8.0), 300 mM NaCl, 1% Triton X-100, 1 mg/mL lysozyme and l x protease inhibitor cocktail [Sigma, #8465]) and underwent 10 cycles of the following: incubation on ice for 5 min, vortexing for 30 s, sonication for 10 s. Cell debris was removed by centrifugation at 14,000 rpm for 30 min at 4 °C, and the supernatant was removed and incubated with HisPur Cobalt resin (Thermo Scientific) while rotating for 30 min at 4 °C to allow for the selective binding of histidine-tagged hnRNP LL. The resin was washed by washing Buffer A (50 mM NaH2P04 with 0.4x protease inhibitor cocktail) and B (50 mM NaH2P04 [pH 8.0] and 100 m NaCl with 0.1 * protease inhibitor cocktail) sequentially, and elution buffer (50 mM NaH2P04 [pH 8.0], 300 mM NaCl with l protease inhibitor cocktail) was used to separate hnRNP LL from resin. 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
(Millipore). Purity of hnRNP LL was confirmed by SYPRO Ruby staining. A Bradford assay was performed to determine the protein concentration.
[0116] 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 [γ-32Ρ]-ΑΤΡ. For competition EMS A, PAGE -purified cold (non-labeled) oligomers and 160 μΜ hnRNP LL as a final concentration were preincubated with 20 mM HEPES (pH 6.8), 100 mM KC1, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1 μg/μL BSA, 0.1% Tween 20, 10% glycerol, and 0.01 μg/μL of poly(dl-dC) for 20 min on ice, and end-labeled Py39WT was added for 5 min.
[0117] To determine the effect of IMC-76 and IMC-48 on binding of hnRNP LL to the i-motif, end-labeled Py39WT was incubated with several concentrations of IMC-76 in a buffer (20 mM MES [pH 6.5], 100 mM KC1, 4 mM MgCl2, 1 mM DTT, 1 μg/μL BSA, 0.1% Tween 20, 10%> glycerol, and 0.01 μg/μL of poly [dl-dC]) for 3 h at room temperature. 160 μΜ hnRNP LL was added and incubated for 10 min at 4 °C. The DNA-protein complex and free DNA were visualized by 6% native PAGE (0.5 x TBE and 1.25% glycerol in gel and running buffer) and phosphorimager scanning.
[0118] SPR Analysis: Surface plasmon resonance analyses were performed on a
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. [0119] Streptavidin (Leinco Technologies, Inc., St. Louis, MO) (SA) was immobilized on a CM5 chip using standard amine coupling. Briefly, carboxy groups on the chip surfaces were activated with an injection of 0.05 M N-hydroxysuccinimide/0.2 M 1- ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride at a flow rate of 10 μί/ηιίη for 7 min SA, diluted in 10 mM NaOAc (pH 5.5), 20 μg/mL, and pulsed over the surface at a flow rate of 10 μΕ/ι ίη until 1750 RU was achieved. Temperature was 25 °C and running buffer was 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% Tween 20. Any remaining active esters were blocked by injecting 1 M ethanolamine (pH 8.5) for 7 min at 10 μΕ/ηιίη. The SA surfaces were washed six times with 1 M NaCl/50 mM NaOH at a flow rate of 20 μΕ/ηιίη for 60 s. Both active and reference flow cells had SA.
[0120] The biotinylated oligomers were resuspended in 10 mM Tris (pH 8.0), 1 mM
EDTA at 100 μΜ, then diluted to 1 μΜ in 20 mM HEPES (pH 7.9), 100 mM KC1, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1 μg/μL 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 nM in the same buffer and injected over the active surface at 10 μΕ/ηιίη until 5 RU was captured.
[0121] hnRNP LL was diluted into running buffer (20 mM HEPES for pH 7.9 and pH
6.8 and 20 mM MES for pH 6.5 were used) together with 100 mM KC1, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1 μg/μL BSA, 0.1% Tween 20, and 10% glycerol. These were then injected over the active and reference flow cells. The dilution range was 0.078-10 nM.
Analysis temperature was 18 °C and flow rate was 50 μΕ/ηιίη. Sample compartment was kept at 10 °C. Association time was 300 s (pH 7.9 and pH 6.8) or 420 s (pH 6.5). Dissociation time was 500 s. Surfaces were regenerated with a 10 s pulse of 20 mM NaOH at a flow rate of 30 μΕ/ηιίη, followed by a stabilization time of 350 s.
[0122] 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 KD.
[0123] Circular Dichroism CD analyses were conducted as previously described.
The i-motif-forming oligomers were synthesized by Euro fins MWG Operon. Py39WT and Py39MutT were diluted to 5 μΜ with a buffer (50 mM MES [pH 6.5], 100 mM KC1, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1 μg/μL 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
[0124] Bromine Footprinting: For the Br2 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 MgCl2, 100 mM KC1, 1 mM DTT, 1 μg/μL BSA, 0.1% Tween 20, 10% glycerol, and 0.02 μg/μL poly [dl-dC]) for 5 min at room temperature.
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.
[0125] siRNA Knockdown Assay: siRNA (ID: SASI HsOl 00171042 and
SASI_Hs01_00171043) targeting hnRNP LL (Sigma) was diluted to 50 nM as a final concentration. As a negative control siRNA, ON-TARGETplus Non-targeting Pool
(Dharmacon) was used. For the untreated control, transfection reagent with media only was used. MCF-7 cells (1.5 104 per well of a 12-well plate) cultured in 10% FBS and 1% penicillin/streptomycin-supplemented RPMI were treated with hnRNP LL siRNA with Fugene HD transfection reagent for 72 h. For determining the knockdown effect of hnRNP LL along with IMC-76 treatment, siRNA of hnRNP LL was transfected for 48 h followed by addition of IMC-76 for 24 h. Total RNA was extracted using an RNeasy purification kit (Qiagen) and quantitated by measuring absorbance at 260 nm. 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 Ct 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.
[0126] 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 104) 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.
[0127] ChIP Assay. Both MCF-7 cells (5 105) and BJAB cells (1 106) were cultured overnight and then treated for an additional 24 h with 0.5 of IMC-76 or 2 μΜ IMC- 48. Treatment with DMSO served as the control. To determine the antagonistic effect of two compounds, MCF-7 cells (3-4 x 105 ) were treated with DMSO or 2μΜ 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 μΜ 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). Cells were treated with formaldehyde (1%) to crosslink proteins to DNA for 13 min at rt. 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. Overnight immunoprecipitation with 4 μg of IgG (Cell Signaling, #2729S), acetyl-histone H3 (Millipore, #06-599), Spl (Cell Signaling, #593 IS), or hnRNP LL (Cell Signaling, #4783S) antibodies at 4 °C was followed by addition of protein G-coupled Dynabeads for 90 min 4 °C. Immunoprecipitants were washed with low salt, high salt, and LiCl immune complex wash buffer. Elution with vortexing for 30 min at rt and reverse crosslinking with 200 mM NaCl at 65 °C overnight were performed sequentially. 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 Tm (data now shown), and double normalizations were performed to obtain data (2"AACt). ACt values were calculated by subtracting Ct values of negative region (Ct-Ct neg) and then ΔΔΟ values were obtained by normalizing to ACt of input (ACt-ACt input).
[0128] Quantitative PCR: To determine if IMC-76 and IMC-48 affect the
transcription level of Spl and hnRNP LL, qPCR was conducted using Rotor-Gene Q
(Qiagen). MCF-7 cells (1.5 x 105) and BJAB cells (3 x 105) were treated with 2 μΜ IMC-76 and IMC-48 for 24 h. 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
immunoprecipitation.
[0129] Immunoprecipitation : For further verification of the specificity of these antibodies with MCF-7 cells, IP experiments were conducted. For Spl IP, nuclei isolated by kit (Sigma, NUC101) were incubated with RIPA buffer (Cell Signaling, #9806) for 15 min on ice. For hnRNP LL IP, whole-cell lysate was used. After homogenization using
QIAshredder (Qiagen), the extract was centrifuged at 14000 rpm at 4 °C for 15 min. The supernatant was precleaned with 50 μΐ, of Protein G-coupled magnetic beads (Invitrogen, 10003D) at 4 °C for 1 h. Protein concentration was determined by Bradford assay, and then the supernatant was diluted to 1 μg/μL for Spl and 12.5 μg/μL for hnRNP LL by lysis buffer. For IP, antibodies for Spl and hnRNP LL were added to reach a 1 : 100 and 1 : 10 dilution, respectively. As a negative control, -0.5-1 μg of IgG, optimized to adjust the heavy chain signal in IP samples of IgG and Spl by western blot, was used. Binding of antibodies was conducted at 4 °C overnight for Spl and 2 h at rt for hnRNP LL. Protein G-coupled magnetic bead with 1% BSA was added and incubated for 1 h to precipitate the immunocomplex. The beads carrying the immunocomplex were washed by lysis buffer three times. To dissociate the immunocomplex, 25 μΐ^ of Laemmli buffer was added and heated at 95 °C for 5 min and subjected to SDS-PAGE (6% or 8%). For western blot analysis, 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.
[0130] The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

Claims

What is Claimed is:
1. A method for treating a clinical condition associated with underexpression of B-cell lymphoma 2 (BCL2) gene, said method comprising administering to a subject in need of such a treatment a compound that increase i-motif structure of BCL2 gene.
2. The method of Claim 1, wherein said clinical condition comprises
neurodegenerative disease, amyotrophic lateral sclerosis (ALS) and macular degeneration.
3. The method of Claim 2, wherein said neurodegenerative disease comprises schizophrenia, Alzheimer's disease and Huntington disease.
4. The method of Claim 1, wherein said compound decreases 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% when the ratio of said compound to BCL2 DNA is about 4: 1.
5. The method of Claim 4, wherein said compound decreases the imino proton area of proton nuclear magnetic resonance (NMR) associated with the flexible hairpin species at pH 6.6 at temperature of about 3 °C to about at least 75% when the ratio of said compound to BCL2 DNA is about 4: 1.
6. The method of Claim 1, wherein said compound increases the imino proton area of proton nuclear magnetic resonance (NMR) associated with said i-motif structure at pH 6.6 at temperature of about 3 °C to about at least 50% when the ratio of said compound to BCL2 DNA is about 4: 1.
7. The method of Claim 1, wherein said compound has a steroid core structure.
8. The method of Claim 7, wherein said compound is of the formula:
Figure imgf000052_0001
wherein
R1 is -ORa;
R2 is H or a carbonyl moiety of the formula -C(=0)-Rb or -C(=0)-Rc-X -C(=0)-
Rb;
R3 is hydrogen or -ORa;
R4 is hydrogen or together with R3 forms R5 is -ORa or -NHRd;
R6 is -CH(NReRf)-Rg or -(C=0)-Rh-NReRf, -CH-(CH3)-R9 wherein R9 is branched
CH3 or linear alkyl with 2 - 7 carbon atoms; or A K Λ R1° wherein R is -H or linear or branched alkyl and n = 1 - 4;
each Ra is independently H, alkyl or a carbonyl moiety of the formula -C(=0)-Rb; each Rb is independently H or alkyl;
each of Rc and Rh is independently alkylene;
Rd is H or -C(=0)-X2;
each of Re and Rf is independently alkyl or optionally substituted aryl, heteroaryl or heterocyclyl, or Re and Rf together with the nitrogen atom to which they are attached to form an optionally subsituted five- or six-membered heterocyclyl;
Rg is alkyl
X1 is -O- or -N(Rb)-; and
X2 is optionally substituted aryl, heteroaryl or heterocyclyl, or -NRbRb.
9. The method of Claim 1, wherein said compound is of the formula:
R7-C(=0)-X3-R8,
wherein
R7 is optionally substituted (heterocyclyl)alkyl;
R8 is optionally substituted cyclyl or aryl, or a steroidal moiety; and
X3 is -O- or -NH-.
10. A method for reducing cell apoptosis associated with B-cell lymphoma 2 (BCL2) gene expression, said method comprising contacting cells with a compound that increase i-motif structure of BCL2 gene to increase BCL2 gene expression thereby reducing cell apoptosis.
11. The method of Claim 10, wherein said compound reduces cell apoptosis by at least 25% compared to the amount of cell apoptosis in the absence of said compound.
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