Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/136—Screening for pharmacological compounds

Definitions

• Breast cancer accounts for nearly one quarter of ail cancer diagnoses and is the principle cause of cancer-related mortality in women worldwide (/, 2).
• treatment selection for breast cancer is based on pathological information and histological grade, and on the expression status of the estrogen (ER).
• ER progesterone
• HER2/neu epidermal growth factor 2
• PR progesterone
• HER2/neu epidermal growth factor 2
• I, 5 overall survival
• expression of ER and/or PR is a good prognostic factor and predictive indicator for benefit from endocrine therapies and although HER2 overexpression connotes adverse prognosis, patients greatly benefit from anti ⁇ HER2 targeted treatments (4, J).
• TNBC triple negative subclass of breast cancers
• Casein kinase- 1 delta (CKL16) and epsilon (CKls) are two highly related serine/threonine kinases known to regulate diverse cellular processes, including circadian rhythm, membrane trafficking and the cytoskeleton, and both have been implicated in cancer (8-11).
• CKL16 casein kinase- 1 delta
• CKls epsilon
• i myristolated CKls Is sufficient to transform mammary epithelial cells in vitro whereas expression of a dominant-negative mutant of CK16 impairs SV40 ⁇ induced mammary
• This invention is directed, In various embodiments, to a method of inhibiting CK16, comprising contacting the CK16 with an effective amount or concentration of a compound of formula (I)
• each R is independently selected hydrogen or (Ci-C6)alkyl, or two R groups bonded to a nitrogen atom together with the nitrogen atom can form a 5-7 membered heierocyclyl optionally further comprising 1 or 2 heteroatoms selected from the group consisting of O, S, and NR'êt wherein R' is hydrogen or (Ci ⁇ €js)a!kyl:
• the invent on is directed to a method of treating cancer, including but not limited to breast cancer, melanoma, glioblastoma, meduUoblastoma. renal, bladder and colon cancers.
• cancers like breast cancer that metastasize to the brain, lung, bone axe also amenable to treatment with our € ⁇ 1 ⁇ inhbitors, as long as both elevated CK15 and ⁇ -catenin dependence are involved in those metastatic diseases.
• the method of treatment comprises administering to a patient afflicted therewith, an effective dose of a compound of formula (!) as described above,
• the invention farther discloses that cancers particularly susceptible to treatment with C 15 inhibitors are those cancers with upregulated expression levels of both CK1 and ⁇ - catenin. Accordingly, in various embodiments the invention provides a method of identifying an anticancer drug effective for treatment of a cancer in a patient, comprising determining a level of C 15 and a level of ⁇ -catenin in the patient and selecting a C 15 inhibitor for a treatment regimen if both the levels are elevated.
• the invention provides a method of treating in a patient a breast cancer, a melanoma, a glioblastoma, a meduiloblasioma, or a renal, bladder or colon cancer, or a cancer that metastasizes to the brain, lung, or bone, comprising determining if elevated CK15 and ⁇ -eatenin levels are present in the patient, and if both levels are elevated, selecting a C 16 inhibitor for a treatment regimen.
• the invention provides a method of treatment of a triple negative subclass of breast, cancers (TNBC). In other embodiments the invention provides a method of treatment of an HER+ breast cancer.
• the methods compose administering an effective dose of a compound of formula (I) to a patient afflicted with the breast cancer.
• CK1S is clinically relevant and effective target for select breast cancer su ty es.
• C CSNKID DNA copy number analyses in invasive breast carcinomas clustered according to CK13 expression (n ⁇ 303), Gene-level copy number estimates (G1STIC2 threshold): -2,-1,0,1,2, representing homozygous deletion, single copy deletion, diploid normal copy, low-level copy number amplification, or high-level copy number amplification are shown.
• D Scatter plot of CSNKID Log 2 mRNA expression vs. Log 2 copy number values (972 breast cancer patients).
• E CK15 and CKls protein expression in indicated breast cancer cell lines and MCF10A mammary epithelial cells.
• F Chemical structure of SR- 3029.
• BCM-4013 PDX tumors independent BMC-4013 PDX tumors.
• G Kaplan-Meier survival curve corresponding to studies shown in (F) (p-value calculated using log-rank test).
• H TUNEL staining on serial sections of vehicle and SR-3029 treated BMC-4013 tumors (representative images are shown) (white line scale ⁇ 2Q0 urn).
• Fig, 4 I Modulation of the Wnt/p-eaterdn pathway is a biom&rker for CK1S activity and inhibition,
• A Wnt pathway genes significantly enriched in CK1 overexpressing human breast tumors (fold ehange>2, p-value ⁇ 0.05) (red is CK15 gene)
• B Effect of SR-3029 (+) or vehicle (-) treatment (18 hr, 30 nM) on nuclear vs. cytoplasmic ⁇ -catenin in the indicated breast cancer cell lines.
• C Expression of active ⁇ -catenin (ABC) in MB A-MB-231 cells after 18 hr treatment: with SR-3029 or vehicle or following transfection with C 16 siRNAs (harvested at 48 hr).
• NT non-targeting
• Fig, 5 I C IS is a necessary md sufficient driver of Wnt/p-eatenm signaling in biimgsi bre&st esneer,
• A Cell growth (left) and apoptosis (right) measured after 72 hr +/- SR-3029 in MD
• C C
• Fig, 6 I OQ ⁇ is i3 ⁇ 4 driver of Wnt/p-catenin signaling in vim.
• A Expression of nuclear and cytoplasmic ⁇ -cate n
• TCGA Frequency of CSNK1D copy number amplifications
• TCGA renal papillary cell carcinoma
• bladder cancer tuniors TCGA
• F -logs op-values showing significant overlap between Wnt p-caienin pathway genes and C I6 signature lists (p ⁇ G.G5 > fold change >! .5) for indicated cancer types (red line is threshold of significance, p-0.05).
• CSNK1D is amplified and/or overexpressed in human breast tuniors and that C- ⁇ is an imexploited vulnerability of human breast cancer subtypes overexpressing this kinase.
• selective knockdown of CK16, or treatment with an -house, highly selective and potent C 15 inhibitor triggers apoptosis of CK16- expressing breast tumor cells ex vivo, potent tumor regression in orthotopic models of triple negative breast cancer, including patient derived xenografts, and tumor growth inhibition in HER2+ breast cancer models.
• C 15 is a promising target for breast cancer therapeutics, and demonstrate the efficacy of an i-house, selective and potent small molecule Inhibitor that is effective against breast cancer subtypes overexpressing CK16. Further, we demonstrate that the gene encoding CK.15, CSNK1D, is frequently amplified and/or overexpressed In a subset of human breast cancers, across each of the major breast cancer subtypes, and that knockdown or inhibition of CK18 provokes breast tumor regression in patient derived xenograft and breast cancer cell line orthotopic xenograft models of TNBC and HER2+ breast cancer. Notably, our mechanistic studies have established that C 18 activity is a critical driver of Wat -catenin pathway activation in breast cancers, a molecular phenotype known to associate with poor prognosis in breast cancer patients.
• Our results are discussed in greater detail below.
• Our invention provides a method of inhibiting casein kinase 1 ⁇ (CKld), and consequently, a method of treating breast cancer, comprising contacting the C 16, e.g., in the tissue of a living patient, with an effective amount of a compound of formula (I), or more specifically a compound of formula SR-3029, the structures of which are shown above.
• CKld casein kinase 1 ⁇
• CSNKlD ⁇ is Amplified and/or Overexpressed in a Subset of Human Breast cancers
• CK15 and C le were assessed the expression of each isoform in human breast tumor specimens compared to normal mammary tissue.
• ixTimunohistochemical analyses confirm overexpression of CK16 in human breast tumor specimens versus normal breast tissue and CK16 was overexpresse across a panel of human breast cancer cell lines (Fig IE).
• high C le expression was detected in only 3 of the breast cancer cell lines analyzed (Fig. I E) and expression of both CKI isoforms was low in immortal human MCFI OA breast epithelial cells as well as in the MCF7 and T47D ER+ breast cancer cells.
• SR-3029 (Fig. IF)
• KCso's in the low nanomolar range 5-70 nM
• MCF7 and T47D breast cancer ceils and the MCFIOA cell line which all expressed low levels of CK18, were 2-3 orders of magnitude less sensitive to SR-3029 (Fig. !G).
• SR-3029 inliibits only 1% of the kinome and has a weaker affinity for only three kinases in addition to CK18 and CKls (CDK4, MYLK4 and FLT3) (10, Of these, only FLT3 is a common (and weak) off-target of SR-3029 and its close analogues SR-2890 and SR-1277, which all show similar anii-proliferative activities against human cancer cells ( ), including MDA-MB-231 TNBC cells.
• SR-3029 exhibits pharmacokinetic properties amenable for in vivo studies (16).
• SR-3029 in preclinical tumor models, MDA-MB-2 1 TNBC cells engineered to express firefly luciferase were engrafted into the mammary fat pads of immunodeficient nude mice and seven days later treatment began.
• SR-3029 (20 mg/kg daily i.p.) markedly impaired tumor growth (Fig, 2A S B), Indeed, only 2 recipients subjected to SR-3029 therapy developed tumors, and these were strikingly small in size compared to those that arose in vehicle treated mice (Fig. 2B, C).
• MDA-MB- 231 cells were engineered to stably express a doxycycline (Dox)-inducible shRNA-directed against CK16 (MDA-MB-23 l-shCK15).
• Dox doxycycline
• Wnt -catenin Signaling is a Hallmark of CK16-Expressmg Breast Cancers
• TCGA patient tumor datasets were analyzed for gene signatures associated with CK15 overexpression. 612 genes were identified whose expression significantly correlated with C 1 expression (fold change>2 s p-value ⁇ 0.05), and Ingenuity Pathway Analysis (IP A) identified significant overlap with genes of the canonical Writ pathway, including Wnt/p-catenin targets (CCND1), Wnt pathway components (FZD.9) and endogenous modulators of the pathway (WNT3, WNT9A and SFRP1) ⁇ Secreted frizzied- elated protein I) (Fig. 4A).
• Wnt/p-catenin targets CCND1
• Wnt pathway components FZD.9A
• SFRP1 endogenous modulators of the pathway
• CK1 S knockdown or SR-3029 treatment led to a reduction in the unphosphorylated, active form of ⁇ -catenin (ABC, Fig, 4C) and markedly reduced endogenous p-catenin TCF transcriptional activity, as measured in MDA-MB-231 cells stably expressing a TCF-dependent lutiferase reporter (Fig. 4D).
• SR-3Q29 repressed the expression of endogenous p- catenin TCF target genes, including CCNDl, MYQ CD44, as well as WNT3 and WNT9A, which were all associated with CK16 expression in human tumors (Fig, 4E, F).
• SR-3029 treatment also resulted in a marked increase in the expression of the endogenous Wnt antagonist SFRP1, yet had no effect on Notch pathway mRNAs (JAG1, NUMB, DTX) (Fig. 4G) > an additional pathway strongly implicated in breast cancer pathogenesis (27).
• ⁇ -eatenin shRNAs in MDA-MB-231 and MDA-MB-468 cells. Each of these cell types expressed high levels of nuclear ⁇ -eatenin (Fig, 4 J) and were dependent on ⁇ -catenin for sustained cell growth and survival (Fig, 4K). Conversely, MCF7 cells, which express little-to-no nuclear ⁇ - catenln, were insensitive to ⁇ -caienm knockdown, consistent with their low expression of CRT ⁇ and relative insensitivity to SR-3029 (Fig. 4 ). To more directly assess the role of impaired ⁇ - catenin signaling in the anti-tumor activity of targeting CK16 S we utilized two constitutively active ⁇ -catemn mutants. Forced expression of or the Ffc-terniinal
• MCF7 ER+ breast cancer cells express low levels of C 15 (Fig, IE), have reduced expression of active (nuclear) ⁇ -catenin versus MDA-MB-231 cells, are refractor ⁇ ' to SR-3029 (Fig. 1G) and have limited tumorigenic potential versus other hnman breast cancer cells (29-31),
• MCF7 cells engineered to overexpress CK15 displayed increased expression of nuclear ⁇ -eatenin (Fig. 5C, D) and downstream Wnt target genes, including CCNDL CD44, WNT3 and WNT9A (Fig.
• MDA-MB-231 tumors isolated from mice treated for 7 days with 20 mg kg SR-3029, or vehicle (once daily, Lp. administration) were analyzed for markers of activated ⁇ -catenin signaling.
• Expression of nuclear ⁇ -catenin was markedly reduced in tumors derived from SR-3029 treated mice compared to vehicle treated controls (Fig, 6 A), and this was associated with decreases in CCNDL WNT3, WNT9A and KARA niRNA transcripts, as well as with a marked increase in expression of SFRP1 transcripts (Fig. 6B).
• the CK!5 ⁇ to ⁇ P ⁇ eatenm signaling circuit may be an unexploited vulnerability across a spectrum of human malignancies. Identification of specific drivers of human breast cancer has instructed the development of targeted therapies such as trastuzumab for the treatment for HER2 amplified breast cancers and hormonal therapies for the treatment of ER+ breast cancers, and these targeted agents have improved the survival and clinical management of these diseases (32), In contrast, patients with relapsed disease and those with TNBC lack targeted therapies, and represent an urgent unmet clinical need. The data presented herein implicates CK16 as a highly attractive therapeutic target with potential benefit for HER2+ and TNBC breast cancer patients aberrantly expressing CK15.
• CK15 which is widespread across each of the four major breast cancer subtypes, may thus identify tumors that will respond to this targeted treatment strategy. Further study across a broader spectrum of patient-derived timior samples is needed to fully investigate this hypothesis.
• Gain- and loss-of-fimction mutations in positive ( ⁇ -catenin) and negative (APC, AXIN1, etc.) regulatory components of the Wnt path way are prevalent at a high frequency in human cancers (revie wed by (34) ⁇ .
• Wnt pathway components in these malignancies are rare (20, 21, 37).
• CK15 o-p-catenin signaling is activated in a subset of human breast cancers, where silencing or pharmacological inhibition of CK18 is sufficient to disable ⁇ -catenin activity and provoke breast cancer cell apoptosis
• Our findings thus implicate CK15 as a key target kinase mat can be exploited (for instance by SR-3029) to disable aberrant Wnt/p-catemn signaling that is manifest in several breast cancer subtypes.
• Previous reports have described several pathways where CK16 plays an important role (23, 33).
• SR-3029 clearly targets Wnt/p-eatenin signaling, additional effectors could contribute to its substantial anti-tumor activity in vivo.
• CK 5 is: (i) activated via amplification and/or overexpression; (ii) a necessary driver of p ⁇ catenin activity in these tumor subtypes; and (Mi) necessary for the growth and survival of cell and preclinical models of human breast cancer.
• the invention can provide a method of inhibiting casein kinase 18 (C 16), comprising contacting the C 16 with an effective amount or concentration of a compound of formula (!
• each R is independently selected hydrogen or (C]-C ⁇ s)alkyl, or two R groups bonded to a nitrogen atom together with the nitrogen atom can form a 5-7 memhered heterocyclyl optionally further comprising 1 or 2 heteroatoms selected from the group consisting of O, S, and N 5 , wherein R' is hydrogen or ⁇ CrC 6 ) !kyl;
• the invention further provides, in various embodiments, a method of treating cancer, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I)
• each R is independently selected hydrogen or (Ci-CeJaLkyl, or two R groups bonded to a nitrogen atom together with the nitrogen atom can form a 5-7 membered heterocyclyi optionally further comprising 1 or 2 heteroatoms selected from the group consisting of O, S, and NR' 3 wherein R' is hydrogen or (Ci-C ⁇ alkyl;
• the cancer that can he treated with an effective amount of a compound of formula (I) can be a cancer wi th upregulated expression levels of both.
• CK16 and ⁇ -catenin can be breast cancer, melanoma, glioblastoma, medulloblastoma, renal, bladder or colon cancer, or can be a cancer that metastasizes to the brain, lung, or bone provided that both elevated CK16 and ⁇ -catenin dependence are involved irt those metastatic diseases.
• the breast cancer can be of the triple negative subclass of breast cancers (TNBC) or is an HER+ breast cancer.
• the invention can provide a compound of formula (I) of claim 1 for treatment of cancer, such as wherein the compound is SR-3G29, or a pharmaceutically acceptable salt thereof.
• the cancer that can be treated with an effective dose of a compound of formula (I) can be a breast cancer, melanoma, glioblastoma, medulloblastoma, renal, bladder or colon cancer, or can be a cancer that metastasizes to the brain ⁇ lung, or bone provided that both elevated C 16 and ⁇ -eatenin dependence are involved in those metastatic diseases.
• the cancer can be a breast cancer of the triple negative subclass of breast cancers (TNBC) or can be an HER+ breast cancer.
• the invention further discloses that cancers particularly susceptible to treatment with C 1 ⁇ inhibitors are those cancers with upregulated expression levels of both C 15 and ⁇ - catemn. Accordingly, the invention provides, in various embodiments, a method of identifying an anticancer drug effective for treatment of a cancer in a patient, comprising determining level of CK16 and a level of P-catenin in the patient and selecting a C 15 inhibitor as the anticancer drag if both the levels are elevated.
• Che invention can provide a method of treating in a patient a breast cancer, a melanoma, a glioblastoma, a medulloblastoma, or a renal, bladder or colon cancer, or a cancer that metastasizes to the brain, lung, or bone, comprising determining if elevated CK16 and ⁇ -eatenin levels are present in the patient, and if both levels are elevated, selecting a C .16 inhibitor for a treatment regimen.
• the level of € ⁇ 1 ⁇ and the level of ⁇ -calenin can be determined using ordinary skill and knowledge in the art in conjunction with the disclosed subject matter herein.
• a level of C 6 or ⁇ -catenin in a patient is considered to be "elevated” if its concentration in body tissue is statistically significantly higher than average levels in comparable patients.
• Treating refers to an. alleviation of symptoms associated with a disorder or disease, or inhibition of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder, or curing the disease or disorder.
• an "effective amount” or a “therapeutically effective amount” of a compound of the invention refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents, or provides prophylaxis for, the disorder or condition
• a “therapeutically effective amount” refers to an amount that is effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result
• a therapeutically effective amount is also one in which any toxic or detrimental effects of compounds of the invention are outweighed by the therapeutically beneficial effects.
• an effective amount when used to describe therapy lo an individual suffering from a disorder, refers to the quantity or concentration of a compound of the invention that is effective to inhibit or otherwise act on ⁇ in the individual's tissues wherein CK16 is involved in the disorder, such as breast cancer . , wherein such inhibition or other action occurs to an extent sufficient to produce a beneficial therapeutic effect,
• phrases such as "under conditions suitable to provide” or “under conditions sufficient to yield” or the like, in the context of methods of synthesis, as used herein refers to reaction conditions, such as time, temperature, solvent, reactant concentrations, and the like, that are within ordinary skill for an experimenter to vary, that provide a useful quantity or yield of a reaction product. It is not necessary that the desired reaction product be the only reaction product or that the starting materials be entirely consumed, provided the desired reaction product can be isolated or otherwise further used.
• Compounds useful in practice of the invention cars be prepared according to literature procedures in conjunction with ordinary knowledge and skill in organic synthesis,
• stable compound and “stable structure” are meant to indicate a compound that Is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.
• each individual integral number representing the number of carbon atoms is intended, For example, recitation of a (Cj -C- alkyl group indicates that the alkyl group can be any of methyl, ethyl, propyl, isopropyl, butyl, sec-huty!, isobutyl, or tert-butyl. It is understood that a specification of a number of carbon atoms must be an integer.
• substituted refers to an organic group as defined herein in which one or more bonds to a hydrogen atom contained therein are replaced by one or more bonds to a non- hydrogen atom such as, but not limited to, a halogen (e.g., F, CI, Br s or I); an oxygen atom in groups such as hydroxy! groups, a!koxy groups, aryloxy groups, aralkyloxy groups,
• oxo(earbonyl) groups carhoxyl groups including carboxylic acids, carboxylates, and carboxylate esters
• a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups
• a nitrogen atom in groups such as amines, hydroxylamines, nitriles, nitro groups, nitroso groups, N-oxides, hydrazides, azides, and enamines
• substituents that can be bonded to a substituted carbon (or other) atom include F.
• R can be hydrogen or a carbon-based moiety, and wherein the carbon-based moiety can itself be further substituted; for example, R can be hydrogen, alkyl, aeyl, cycloaikyl, aryl, aralkyl, heterocyely!, heteroaryl, or heteroarylalkyl, wherein any alkyl, acyl, cycloaikyl, aryl, aralkyl, heterocyelyl, heteroaryl, or heteroarylalkyl can be further independently mono- or multi-substituted with some or all of the above-listed functional groups, or with other functional groups; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heteroeyelyi which can be further mono- or independently multi- sub
• a substituent can be halo, nitro, cyano, OR, ⁇ 1 ⁇ 2, or R, or is C(0)QR, C(0) R 2 , OC(0)OR, OC(0)NR 2 , N(R)C(0)OR, N(R)C(0)NR 2 or thio/thiono analogs thereof.
• thio/thiono analogs thereof with respect to a group containing an 0, is meant that any or all O atoms in the group can be replaced by an S atom; e.g., for group C(0)OR, a
• thio/thiono analog thereof includes C(S)OR, C(0)SR, and C(S)SR; e.g., for group GC(G)NR 2 , a "thio/thiono analog thereof includes SC(Q)MR 2 , OC(S)NRa ⁇ and 5C(S)NR 2 ; and so forth, in various embodiments, a substituent can be any of halo, (Cl-C6)alkyl, (Cl ⁇ C6)alkoxy, (Cl-C6)haloalkyl ⁇ hydroxy(Cl-C6)alkyl s alkoxy(Cl-C6)alkyl s (Cl-C6)alkanoyl, (Cl- C6)alkanoyioxy, cyano, nitro, azido, R 2 N, R 2 NC(0), R 2 NC(0)0 ; R 2 NC(0)NR, (Cl-C6)alkenyl, (Ci-C6)alkynyl, (C6-CiO)ary
• Alky! groups include straight chain and branched carbon-based groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms, or from 1 to 4 carbon atoms.
• straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups.
• branched alkyl groups include, but are not limited to, isopropyl, iso-bntyl, sec-butyl, t-butyi, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.
• the terai "alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl.
• halo or halogen or “haMe” by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine.
• fluoroalky includes mono- and poly-fluoro alkyl groups; examples include CF ' 3 , C2Fs, and the like.
• alkoxy or "alkoxy! refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined above.
• linear alkoxy groups include but are not limited to methoxy, ethoxy, n-propoxy, n-butoxy, n-pentyloxy, n-hexyloxy, and the like.
• branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert- butoxy, isopentyloxy, isohexyloxy. and the like.
• Exemplar alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.
• Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring.
• An aromatic compound as is well-known in the art, is a multiply-unsaturated cyclic system that contains 4n+2 ⁇ electrons where n is an integer.
• aryl groups include monocyclic and polycyclic ring systems. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups, Aryl groups can be unsubstituted or substituted, as defined above.
• Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed above.
• Heterocyclyl groups or the term "heterocyclyl” includes aromatic and non-aromatic ring compounds containing 3 or more ring members, of which one or more ring atom is a heteroatom such as, but not limited to, N, O, and S.
• a heterocyclyl can be a cycloheieroalkyl, or a heteroaryl, or if polycyclic, any combination tliereof.
• heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members.
• Heteroaryl groups are heterocyclic aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members.
• a heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure, which is a multiply- unsaturated cyclic system that contains 4n+2 ⁇ electrons wherein n is an integer.
• pharmaceutically-acceptable salt refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, purification
• “Pharmaceutically or pharmacologically acceptable” include molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
• preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards,
• Any compound found to be an effective inhibitor of CK15 can likewise be tested in animal models and in human clinical studies using the skill and experience of the investigator to guide the selection of dosages and treatment regimens, e.g., for treatment of breast cancer.
• mice were euthanized when moribund, and/or when tumors became ulcerated or reached greater than 1.2 cm 3 . All cell-based assays were performed In triplicate and repeated at least 3 times, enogra tlm ⁇ Imaging of Mice.
• xenograft tumor fragments ( ⁇ mm J ) were transplanted into the cleared mammary fat pad of recipient SCID/Bg mice (Charles River Laboratories), Mice were treated with SR-3029 or vehicle (10:10:80, DMSO:Tweeo-80:Water) at 20 mg kg daily by Lp. injection.
• Tumor volumes were measured as the indicated intervals using calipers or by luminescence imaging using the IVIS 100 imager (exposure time, 1-60 sec; binning 8; field of view 15 -cm; f/stop 1; open filter) following subcutaneous injection of l ciferin (15 rng/ml, Goldbio Technology). Average radiance (p/s/em 2 /sr) was determined from tumor region-of- interest (ROI) using Living-Image (Xenogen) analysis software.
• Cell proliferation was measured 72 hr after SR-3029 or vehicle treatment using Cell-Tlter Glo (Promega) according to the manufacturers' instructions. ECso values were determined using non-linear regression and a four-parameter algorithm (GraphPad PrismS). For clonogenic assays, cells were plated in 6- well dishes in triplicate at a density of 500-1000 cells per well. After overnight incubation, SR-3029 or vehicle (DMSO) was added to media for 72 hr and cells were allowed to grow out for 7-10 days, dining wixich media was changed every 2-3 days in the absence of compound.
• DMSO DMSO
• Colonies were fixed in 4% paraformaldehyde/PBS and stained with 0,5% methylene blue in 50% ethanol for 1 hr at room temperature and de-stained with water. Colonies with greater than 50 cells were counted using a low magnification light microscope.
• siRNA duplexes were prepared according to the manuiacturer's instructions (Qiagen) and specific knockdown conditions were optimized using the HiPerfeet transfection reagent (Qiagen). A final concentration of 20 xiM total siRNA was used to achieve knockdown. FuGene6 (Roche) was used for D A transfections as per manufacturer's instructions.
• Lentiviral vectors expressing CK18 (Y3989-Lv 105-0200, GeneCopoeia), GFP (EGFP- Lvl 05-0200, GeneCopoeia), luciferase or the TCP reporter 7TFP (Addgene, Roel Musse) were co-transfected with pPAC Hl packaging p!asmids into HEK293T cells to produce lentiviral particles per the manufacturers' recommendations (System Biosciences), To stably express specific shRNAs shRNA oligonucleotides were cloned into the Tet-pLKO-P ro vector using the recommended protocol (38) and lenti viruses were generated using the Mission Packaging System (Sigma).
• MDA-MB-231 cells were transduced with optimized titers of lentiviruses and infected cells were selected in puromycin (1 ⁇ / ⁇ ) or hlasticidin (5 g ml for luciferase lentivirus) containing medium to expand stably infected pools.
• SDS-PAGE gel electrophoresis was performed using NuPAGE 4-12% Bis ⁇ Tris gels (Invitrogen) and transferred to PVDF membranes by semi-dry transfer using trans-biot transfer medium (Biorad). Membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences) and incubated overnight at 4°C with primary antibodies. After repeated washes with TEST (20 mM Tris, pH 7.6, 140 mM NaCl and 0.1% Tween-20) blots were incubated with the appropriate IRDye-eonjugated secondary antibody (LI-COR Biosciences) and imaged using the LI-COR Odyssey.
• TEST 20 mM Tris, pH 7.6, 140 mM NaCl and 0.1% Tween-20
• Total R A was obtained using the RNeasy Plus Mini Kit ( ' Qiagen) and 1-2 ug of RNA was reverse transcribed with Superscript III First Strand Synthesis System (Life Technologies). Quantitative FCR was performed using the Power S YBR Green PGR Master Mix (Life Technologies).
• Intron-spanning gene-specific primer pairs were designed using the Primer3 a!goridmi and relative expression values for each gene of Interest obtained by normalizing to GAPDH mRNA expression using the && C t method, For apoptotic detection in cryosections tumors and small intestines were fixed in 10% buffered formalin for 2 hr, incubated in 20% sucrose overnight and embedded in OCT. Frozen sections (5 ⁇ ) were mounted and stained using the ApopTag Red In situ Kit according to the manufacturer instructions (CHEMl ' CON). For H&E staining, tissues were fixed in 10% buffered formalin for 48 hr, transferred to 70% ethanol PBS and embedded in paraffin. Staining was performed on 5 ⁇ sections following deparaffinization (AMI, Laboratories),
• MDA-MB-231 or HEK293 cells stably expressing the TCF luciferase reporter 7TFP (39) were transfected with p ⁇ eatemn ⁇ S33Y or empty vector (pcDNA). After 18 hr cells were seeded onto a 96-well plate at a density of 6000 cells/well. After 24 hr cells were treated with SR-3029 or vehicle and incubated for 6 hr prior to addition of 1 ⁇ 1 recombinant human Wnt3a (R&D Systems) or PBS, After 3 hr, reporter assays were performed using BriteLite Pius (Perkin Elmer), which was added in equal volume directly to the media and luminescence read using a Spectramax plate reader (Molecular Devices).
• TCGA Cancer Genome Atlas
• BRCA Breast cancer
• KJRC kidney renal papillary cell carcinoma
• BLCA bladder cancer
• RNA-Sequencing R A-Seq
• RSEM Expeetation- Maximization
• CK13 the upper 100 and lower 100 tumor breast tumor samples (CKlS-hig arjd CKldAow groups) or upper and lower quaitiles for smaller datasets (kidney and bladder cancer) were defined based on CK13 (CSNKID) expression.
• CK13 CK13
• GeneSpring Volcano Plot function was used to identify differentially expressed genes between the CKJS- igfr and CKlS-low groups.
• Statistical test parameters were set as follows: selected test, unpaired ? est; p-va!ue computation. Asymptotic, multiple testing correction, Benjamini-Hochberg. Corrected p- value cut-off was set to 0,05 and fold change cutoff was as indicated in the text,
• Virshup, iC261 induces eel! cycle arrest and apoptosis of human cancer cells via
• V Bryja, G. Schulte. E, Arenas, Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1 -mediated phosphorylation of Dvl to activate beta-catenin.
• U. nippsc ild, M K uger, J. Richter, P. X s B, Garcia-Reyes, C, Peifer * J. Halekolte, V, Bakulev, J. Bischof, The C 1 Family: Contribution to Cellular Stress Response and Its Role in Carcinogenesis, Front Oncol 4, 96 (2014)10,3389/fonc.20.4.00096).
• Beta-catenin a novel prognostic marker for breast cancer: its roles in c clin Dl expression and cancer progression. Proc Nad Acad Sci USA 97, 4262-4266 (2000); published online EpubApr 1 1 (10.1073/pnas.060025397).
• GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy- number alteration in human cancers.

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