Patent Application
20180244719
This invention relates to treatment of proliferative disorders. More specifically, the present invention provides therapeutic methods and compositions for treating cancers, such as cancers constitutively expressing STAT3.
The Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that regulates many biological processes including cell proliferation, differentiation and survival (Debnath, et al., Small Molecule Inhibitors of Signal Transducer and Activator of Transcription 3 (Stat3) Protein. Journal of Medicinal Chemistry 2012, 55, 6645-6668; Masciocchi, et al., Signal transducer and activator of transcription 3 (STAT3): a promising target for anticancer therapy. Future Medicinal Chemistry 2011, 3, 567-597; Lavecchia, et al., Novel inhibitors of signal transducer and activator of transcription 3 signaling pathway: an update on the recent patent literature. Expert Opin Ther Pat 2014, 24, 383-400; Page, et al., Signal transducer and activator of transcription 3 inhibitors: a patent review. Expert Opin Ther Pat 2011, 21, 65-83). Under normal physiological conditions, the activation of STAT3 is transient and tightly regulated, and is only triggered by the stimulation of extracellular cytokines and growth factors such as IL-6, EGF and PDGF, which leads to the phosphorylation of a specific tyrosine (Y-705) on STAT3 (Sun, et al., Cucurbitacin Q: a selective STAT3 activation inhibitor with potent antitumor activity. Oncogene 2005, 24, 3236-3245; Turkson, et al., Novel peptidomimetic inhibitors of Stat3 signaling and oncogenesis. European Journal of Cancer 2002, 38, S98-S98). This phosphorylation subsequently induces the dimerization of STAT3-STAT3 which is stabilized by two reciprocal phosphotyrosine-SH2 binding interactions. The phosphorylated STAT3 dimers translocate to the cell nucleus and bind to promoter regions in DNA, resulting in regulation of specific gene expression (Zhang, et al., A Novel Inhibitor of STAT3 Homodimerization Selectively Suppresses STAT3 Activity and Malignant Transformation. Cancer Research 2013, 73, 1922-1933; Zhang, et al., Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts. Proceedings of the National Academy of Sciences of the United States of America 2012, 109, 9623-9628). However, STAT3 is constitutively activated in a variety of cancers including both solid tumors (i.e. breast, prostate, lung, pancreatic) and hematological cancers (i.e. lymphoma, leukemia, melanoma) (Urlam, et al., Development of new N-arylbenzamides as STAT3 dimerization inhibitors. Medchemcomm 2013, 4, 932-941; Siddiquee, et al., An Oxazole-Based Small-Molecule Stat3 Inhibitor Modulates Stat3 Stability and Processing and Induces Antitumor Cell Effects (vol 2, pg 787, 2007). Acs Chemical Biology 2009, 4, 309-309; Cheng, et al., Stat3 Inhibition Augments the Immunogenicity of B-cell Lymphoma Cells, Leading to Effective Antitumor Immunity. Cancer Research 2012, 72, 4440-4448). Such hyper-activation of STAT3 leads to uncontrolled cell proliferation by activating cell cycle regulators such as c-Myc and cyclin D1, and enhancement of cell survival by selectively inducing the expression of anti-apoptotic proteins including Bcl-xL and survivin. As such, STAT3 mediated signaling pathways are recognized as valid cancer targets.
Many approaches have been adopted to inhibit constitutive activation of STAT3. Among the domains of STAT3 that regulate its function are SH2 domain (dimerization domain) and the DNA-binding domain, seen in
The exploration of new and non-covalent molecular ligands that selectively inhibit STAT3-DNA binding are therefore very significant, as such an effort will not only lead to novel anti-cancer therapeutics, but also provide a new tool to further dissect the functional role of STAT3 in the regulation of cell proliferation and apoptosis. However, the art is currently underdeveloped in this field. As such, novel STATS-DNA binding inhibitors are required for therapeutic and academic use.
Based on chiral PNA backbone, a new class of peptidomimetics termed “γ-AA peptides” were recently developed, as seen in
For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:
As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes a mixture of two or more polypeptides and the like.
As used herein, “about” means approximately or nearly and in the context of a numerical value or range set forth means ±15% of the numerical.
As used herein “animal” means a multicellular, eukaryotic organism classified in the kingdom Animalia or Metazoa. The term includes, but is not limited to, mammals. Non-limiting examples include rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Wherein the terms “animal” or “mammal” or their plurals are used, it is contemplated that it also applies to any animals.
As used herein, the term “proliferative disorders” broadly encompasses any neoplastic disease(s) including those which are potentially malignant (pre-cancerous) or malignant (cancerous) and covers the physiological condition in mammals that is typically characterized by unregulated cell growth. The term therefore encompasses the treatment of tumours. Examples of such proliferative disorders include cancers such as carcinoma, lymphoma, blastoma, sarcoma, and leukemia, as well as other cancers disclosed herein. The compositions disclosed herein are useful for treating all types of cancer, and in particular cancers which express STAT3. Cancers having constitutively expressed STAT3 include breast cancer; ovarian cancer, multiple myeloma tumor specimens, pancreatic cancer and blood malignancies, such as acute myelogenous leukemia, (Turkson, et al., U.S. Pat. No. 8,609,639; Jove, et al., WO 00/44774), multiple myeloma, acute myelogenous leukemia (Dalton, et al., PCT/US2000/001845), head and neck cancer, lung cancer, colorectal carcinoma, prostate cancer, melanoma, sarcoma, liver cancer, brain tumors, multiple myeloma, leukemia, cervical cancer, colorectal carcinoma, liver cancer, gastric cancer, prostate cancer, renal cell carcinoma, hepatocellular carcinomas, gastric cancers, and lymphomas (Li, et al., U.S. application Ser. No. 12/677,513), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, a seminoma, an embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, a glioma, an astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma; acute lymphocytic leukemia, acute myelocytic leukemia, chronic leukemia, and polycythemia vera (Jove, et al., U.S. application Ser. No. 10/383,707).
As used herein the term “patient” means members of the animal kingdom, including mammals, such as but not limited to, primates including humans, gorillas and monkeys; rodents, such as mice, fish, reptiles and birds. The patient may be any animal requiring therapy, treatment, or prophylaxis, or any animal suspected of requiring therapy, treatment, or prophylaxis. The term treatment, as used in this definition only, is intended to mean that regiment described is continued until the underlying disease is resolved, whereas therapy requires that the regiment alleviate one or more symptoms of the underlying disease. Prophylaxis means that regiment is undertaken to prevent a possible occurrence, such as where a pre-cancerous lesion is identified.
As used herein, the term “therapeutically effective amount” refers to that amount of a therapy (e.g., a chemotherapeutic agent) sufficient to result in the amelioration of cancer or other proliferative disorders or one or more symptoms thereof, prevent advancement of cancer or other proliferative disorder, or cause regression of cancer or other proliferative disorder.
All Fmoc protected α-amino acids and Rink amide resin (0.7 mmol/g, 200-400 mesh) were purchased from Chem-Impex International, Inc. TentaGel MB NH2 resin (0.3 mmol/g, 140-170 μm) was purchased from RaPP Polymere GmbH. Masses of γ-AA peptides were obtained on an Applied Biosystems 4700 Proteomics Analyzer. MS/MS analysis was carried out with a Thermo LTQ Orbitrap XL. Solid phase synthesis was conducted in peptide synthesis vessels on a Burrell Wrist-Action shaker. γ-AA peptides were analyzed and purified on a Waters Breeze 2 HPLC system, and then lyophilized on a Labcono lyophilizer. All cell lines were obtained from ATCC (American Type Culture Collection, Manassas, Va., USA). Human breast carcinoma MDA-MB-468 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS), 10 mM sodium pyruvate, 25 mM HEPES, pH 7.5, 1000 U/ml penicillin, and 1000 μg/ml streptomycin. Primary antibodies against pY705-STAT3, Cyclin D1 and Survivin were purchased from Cell Signaling Technology (Danvers, Mass.). Primary antibodies against STATS (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Primary antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, Mo.).
The OBOC γ-AA peptide library was synthesized as provided in
On-bead Screening of the γ-AA peptide library was performed using STAT3 as a target for the combinatorial library screen. The synthesized library compounds were stored in a peptide synthesis vessel, and then washed and incubated in the same container. The beads were screened and picked up manually under Zeiss inverted fluorescence microscope 10×43HE filter. In order to avoid any possible nonspecific binding, both the STAT3 and antibodies solution were made in 1% BSA/TBST blocking buffer.
The library synthesized on TentaGel beads (1.6 g, 800,000 beads, 160,000 compounds) as discussed previously. The beads were then swelled in DMF for 1 h, washed with 1×TBST five times and then equilibrated in 1×TBST overnight at room temperature. The beads were blocked in 1% BSA in TBST with a 1000× excess of cleared E. coli lysate for 1 hour, washed and equilibrated in 1×PBST before prescreening and screening.
To prescreen, the beads were incubated with mouse 1:1000 diluted STAT3 anti-mouse IgG primary antibody for 2 hours at room temperature, followed by five times 1×PBST wash and incubation with 1:1000 diluted goat anti-mouse IgG conjugated with Alexa Fluor dylight 594 for 2 hours. The beads were washed with 1×PBST completely and transferred into a 6-well plate, and the bright red beads were picked up under bench-top microscope for they had suspicious nonspecific binding. The rest of the beads were pulled together, washed with 1×PBST (5×), and then treated with 1% SDS at 90° C. for ten minutes to remove any bound proteins. Then the beads were washed with both water (5×) and 1×TBST (5×) to wash away the SDS and swelled in DMF for 1 hour. After washing (5×) and equilibrating in 1×TBST overnight, the beads were ready for actual STAT3 screening.
The prescreened beads were equilibrated in 1% BSA/PBST for 1 hour at room temperature. After washing with 1×PBST (3×), the beads were incubated with STAT3 peptide at a concentration of 20 μg/mL for 4 hours at room temperature with a 1000× excess of E. coli lysate. After thoroughly washing with 1×PBST, the library beads were incubated in 5 mL of 1% BSA/PBST containing 1:1000 diluted STAT3 anti-mouse IgG primary antibodies for 2 hours at room temperature. The beads were gently washed with 1×PBST (3×) and incubated with 1:1000 diluted goat anti-mouse IgG conjugated with Alexa Fluor dylight 594 for 2 hour at room temperature. The beads were washed with 1×PBST and transferred into the 6-well plate to be observed under Zeiss inverted fluorescence microscope equipped with a 10×43HE filter. Again the individual bright red beads were picked out manually using pipette tips as candidates for further study.
The putative beads were collected and washed with 1×PBST three times. The bound fluorescent dyes, proteins, and antibodies were removed by treating beads with 1% SDS solution at 90° C. for 10 min. After washing with water (3×), DMSO (3×), and acetonitrile (5×), beads were then subjected to CNBr treatment (50 mg CNBr in 1 mL 5:4:1 acetonitrile:acetic acid:H2O) to cleave the compound from the beads for decoding using a previous procedure. The MALDI-MS was run on a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Higher Energy Collision Dissociation (HCD) was performed at collision energy of 25 or 35 mV. HCD fragmentation of a double charged precursor ion was represented in
A screening protocol was then carried out to identify ligands that potentially target STAT3-DNA binding. In brief, the library was first incubated with STAT3 (full length STAT3 protein) (please see experimental for details), followed by the incubation with anti-STAT3 antibody, as seen in
Compounds 1-4 were resynthesized on rink amide resin following a previously reported procedure, as shown in
In an effort to test the ability of the lead γ-AA peptides to inhibit STAT3-STAT3 dimerization, fluorescence polarization assays were conducted to determine whether these molecules disrupt the binding of STAT3 to fluorescein-labelled GpYLPQTV (SEQ ID NO: 1) phosphotyrosine peptide which is known to bind the STAT3-SH2 domain (Zhang, et al., A Novel Inhibitor of STAT3 Homodimerization Selectively Suppresses STAT3 Activity and Malignant Transformation. Cancer Research 2013, 73, 1922-1933). None of these molecules show any inhibitory activity, as seen in
To assess if these γ-AA peptides bind to STAT3 and therefore inhibit STAT3-DNA binding, an in vitro STAT3 filter assay was carried out, as seen in
The SH2-binding phosphotyrosine peptide, GpYLPQTV (SEQ ID NO: 1), (IC50 of 150 nM for inhibition of dimerization of STAT3-STAT3 in vitro in FP assays; Ren, et al., Identification of a high-affinity phosphopeptide inhibitor of Stat3. Bioorganic & Medicinal Chemistry Letters 2003, 13, 633-636) inhibited STAT3-DNA binding by 40% and 60% at 10 μM and 100 μM, respectively, in this STAT3 filter assay, as seen in
The γ-AA peptides were tested to determine if the compounds can pass through cell membranes and retain STAT3-DNA binding inhibitory activity in whole cells. As phosphotyrosine peptide GpYLPQTV (SEQ ID NO: 1) is not cell permeable, a previously reported small molecular inhibitor of STAT3 dimerization, S3I-1757, shown in
To assess the ability of lead γ-AA peptides to modulate the expression of STAT3 regulated genes, Western immunoblotting was carried out to determine the effect of γ-AA peptides on the expression levels of survivin and cyclin D1. Briefly, Western blots were prepared using cells that were harvested and lysed for 30 min on ice with occasional vortexing in 150 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol, 5 mM NaF, 1 mM DTT, 1 mM PMSF, 2 mM Na3VO4, and 5 μg/ml leupeptin. Proteins readings were done using the Bradford protein assay, and equal amounts of protein for each sample were loaded into the wells of SDS-PAGE gels. After separation, proteins were transferred to nitrocellulose and Western blots were performed as previously described (Zhang, et al., A Novel Inhibitor of STAT3 Homodimerization Selectively Suppresses STAT3 Activity and Malignant Transformation. Cancer Research 2013, 73, 1922-1933).
Consistent with FP results, none of γ-AA peptides inhibited STAT3 phosphorylation, while S3I-1757, blocked STAT3 phosphorylation as expected as seen in
To rationalize the findings that these γ-AA peptides can disrupt STAT3/DNA binding, a computer molecular modeling was carried out by docking the most effective inhibitor 1 onto the STAT3 domain that binds DNA. The docking of the γ-AA peptide on the STAT3 DNA-binding domain was performed with Glide (Schrodinger) program. The crystal structure (PDB 1BG1) of STAT3 was used for docking. The structure was prepossessed with protein preparation wizard and then the energy minimization was applied to the structure. A box size of 20 Å×20 Å×20 Å that covers DNA STAT3 binding interface was used as the grid. The ligands were applied with a conformation search, and these conformations were used to dock the STAT3 DNA binding domain.
γ-AA peptide 1, containing multiple negatively charged carboxylate groups, is highly complementary to the STAT3 binding domain in which many cationic and polar amino acid residues are present. The three carboxylate groups interact with positively charged residues R423, R382 and K340, respectively, through electrostatic attraction, which may account for the most critical force for the binding affinity of 1 towards STAT3-DNA-binding domain. In addition, the phenyl ring near the N-terminus inserts deeply into the hydrophobic pocket formed by L430, I431 and V432. The hydrophobic interaction may further contribute to the binding specificity and affinity. Furthermore, the backbone of 1, including its C-terminus, made a few contacts with other polar and charged residues including E415, R417, N466 and Q409. Overall, the modeling suggests that the STAT3-DNA binding domain is highly positively charged. As the most negatively charged sequence in the identified γ-AA peptides, 1 binds to STAT3-DNA binding domain through a range of charge-charge interactions and hydrophobic interactions. Interestingly, the least potent γ-AA-peptide 4 has only one carboxylate that is not optimally positioned to interact with residue K340. The modeling also provides some insights into future rational design of molecules for the inhibition of STAT3-DNA binding.
An OBOC combinatorial γ-AA peptide was developed, which led to successful identification of lead compounds that disrupt STAT3-DNA interaction in nuclear extracts. The fact that these γ-AA peptides do not inhibit the binding of GpYLPQTV (SEQ ID NO: 1) to STAT3 distinguishes them from STAT3-STAT3 dimerization inhibitors (REFs). Furthermore, despite their fairly large size, the γ-AA peptides were taken up by human cancer cells, and inhibited STAT3-DNA binding and STAT3-regulated gene expression. This is not only the first report of γ-AA peptides inhibiting STAT3 function but also that γ-AA peptides are among the first few molecules that bind to STAT3 DNA-binding domain non-covalently and disrupt STAT3-DNA interaction. The results herein strongly suggest that STAT3 DNA-binding domain is a novel target for inhibiting STAT3 function, for use as novel anti-cancer agents targeting STAT3 signaling. In addition, the γ-AA peptide OBOC library can be used to identify chemical probes or drug candidates against targets traditionally believed “undruggable”. Thus, with appropriate modification and further development of γ-AA peptide libraries, this strategy could be employed to develop more potent and specific ligands that bind to a variety of medicinally relevant targets.
In the preceding specification, all documents, acts, or information disclosed does not constitute an admission that the document, act, or information of any combination thereof was publicly available, known to the public, part of the general knowledge in the art, or was known to be relevant to solve any problem at the time of priority.
The disclosures of all publications cited above are expressly incorporated herein by reference, each in its entirety, to the same extent as if each were incorporated by reference individually.
While there has been described and illustrated specific embodiments of the invention, it will be apparent to those skilled in the art that variations and modifications are possible without deviating from the broad spirit and principle of the present invention. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.
This application is a divisional of and claims priority to currently pending U.S. application Ser. No. 15/332,402 entitled “γ-AA-peptide STAT3/DNA Inhibitors and Methods of Use”, filed Oct. 24, 2016, which is a continuation of and claims priority to International Patent Application No. PCT/US2015/027801, filed Apr. 27, 2015 which claims priority to U.S. Provisional Application No. 61/984,179 entitled “Identification of Novel Inhibitors that Disrupt STAT3/DNA Interaction from γ-AA-peptide OBOC Combinatorial Library”, filed Apr. 25, 2014 the contents of each of which are hereby incorporated by reference into this disclosure.
This invention was made with Government support under Grant No. RO1 CA140681-05 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61984179 | Apr 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15332402 | Oct 2016 | US |
| Child | 15963454 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2015/027801 | Apr 2015 | US |
| Child | 15332402 | US |
