WO2017023866A1 - Method of targeting stat3 and other non-druggable proteins - Google Patents

Method of targeting stat3 and other non-druggable proteins Download PDF

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WO2017023866A1
WO2017023866A1 PCT/US2016/045043 US2016045043W WO2017023866A1 WO 2017023866 A1 WO2017023866 A1 WO 2017023866A1 US 2016045043 W US2016045043 W US 2016045043W WO 2017023866 A1 WO2017023866 A1 WO 2017023866A1
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stat3
cancer
binding
method
amino acid
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PCT/US2016/045043
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French (fr)
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Chiang Jia Li
Ao YANG
Harry Rogoff
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Boston Biomedical, Inc.
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702Regulators; Modulating activity
    • C07K14/4705Regulators; Modulating activity stimulating, promoting or activating activity
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

The present disclosure provides methods of targeting STAT3 activity by binding one or more amino acid residues in STAT3's hinge pocket which reduces STAT3's binding affinity for a STAT3 DNA binding site. This discovery therefore provides an innovative approach for targeting other previously non-druggable proteins through their linker regions rather than through their catalytic or regulatory domains.

Description

METHOD OF TARGETING STAT3 AND OTHER NON-DRUGGABLE PROTEINS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Patent Application No. 62/199,874, filed on July 31, 2015. The aforementioned application is hereby incorporated herein by reference in its entirety.

Transcription factors, encoded by approximately 10% of genes in the genome, are vital regulators of many cellular processes, and their dysregulations are responsible for numerous human diseases, particularly cancer. However, other than nuclear receptors, transcription factors, like 80% of proteins encoded by our genome, are considered“non-druggable” since they are not enzymes and do not have an active site that can be used to design a therapeutic ligand. Although transcription factors function through binding to their protein and/or nucleic acid partners, the large macromolecular interaction interfaces in the DNA-binding and transactivation domains have proven challenging for designing small molecules that directly block binding between these macromolecules with sufficient potency and selectivity for therapeutic applications. Hence, there is a need in the art for drugs that target the activity of transcription factors implicated in human disease, for example, cancer.

The Signal Transducer and Activator of Transcription 3 (also known as Acute- Phase Response Factor, APRF, DNA-Binding Protein APRF, ADMIO 3, HIES; referred to herein as STAT3) is a member of a family of seven ubiquitous transcription factors, STAT1 to STAT6, including STAT5a and STAT5b that function at the junction of several cytokine-signaling pathways. For example, STATs can be activated by receptor associated tyrosine kinases like Janus kinases (JAKs) or by receptors with intrinsic tyrosine kinase activity such as, for example, PDGFR, EGFR, FLT3, EGFR, ABL, KDR, c-MET or HER2. Upon tyrosine phosphorylation by receptor associated kinases, the phosphorylated STAT protein (“pSTAT”) dimerizes, as a homo- or heterodimer, and translocates from the cytoplasm to the nucleus, where it binds to specific DNA-response elements in the promoters of target genes and induces gene expression. STAT 2, 4, & 6 regulate primarily immune responses, while STAT3, along with STAT1 and STAT5, regulate the expression of genes controlling cell cycle (CYCLIN D1, D2, and c-MYC), cell survival (BCL-XL, BCL-2, MCL-1), and angiogenesis (HIF1α, VEGF) (Furqan et al. Journal of Hematology & Oncology (2013) 6:90). STAT3 is therefore a prime example of a human transcription factor that is considered a highly compelling target for therapeutic intervention because of its involvement in a variety of human diseases, including malignant, inflammatory, and autoimmune disorders.

In normal cells, STAT3 activation is transient and tightly regulated, lasting for example, from about 30 minutes to a few hours. However, in a wide variety of human cancers, including all of the major carcinomas as well as some hematologic tumors, STAT3 is found to be aberrantly active. Persistently active STAT3 is present in more than half of all breast and lung cancers as well as colorectal cancers (CRC), ovarian cancers, hepatocellular carcinomas, and multiple myelomas and in more than 95% of all head/neck cancers. STAT3 is a potent transcription regulator that targets a large number of genes involved in cell cycle, cell survival, oncogenesis, tumor invasion, and metastasis, including, but limited to, BCL-XL, c-MYC, CYCLIN D1, IDO1, PDL1, VEGF, MMP-2, and SURVIVIN. The collective expression of these STAT3 responsive genes maintains the stemness of cancer stem cells (CSCs) required for the survival and propagation of cancer stem cells. As used herein, "stemness" generally means the capacity for a stem cell population to self-renew and transform into cancer stem cells (Gupta PB et al., Nat. Med. 2009; 15(9):1010-1012). While CSCs form only a small percentage of the total cancer cell population in a tumor (Clarke MF, Biol. Blood Marrow Transplant. 2009; 11(2 suppl. 2):14-16), they give rise to heterogeneous lineages of differentiated cancer cells that make up the bulk of the tumor (see Gupta et al. 2009). In addition, CSCs possess the ability to spread to other sites in the body by metastasis where they seed the growth of new tumors (Jordan CT et al. N. Engl. J. Med.2006; 355(12):1253-1261).

The induction and maintenance of stemness properties in CSCs is fueled by a progressive dysregulation of stemness signaling pathways including, but are not limited to, those signaling pathways associated with Janus kinase/ signal transducers and activators of transcription (JAK/STAT), Hedgehog (Desert (DHH), Indian (IHH), and Sonic (SHH))/PATCHED/(PTCH1)/ SMOOTHENED (SMO), NOTCH/DELTA-LIKE (DLL1, DLL3, DLL4)/JAGGED (JAG1, JAG2)/ CSL (CBF1/Su(H)/Lag-1), WNT/APC/GSK3/β-CATENIN/TCF4 and NANOG (Boman BM et al., J. Clin. Oncol. 2008; 26(17):2828-2838).

It is the aberrant regulation of these stemness signaling pathways in CSCs (see Boman et al.2008) that is presumed to confer resistance to chemotherapy and radiation treatment in CSCs which eventually leads to the relapse and spread of the cancer. Thus, while chemotherapy and radiation kills the majority of rapidly dividing bulk cancer cells in a tumor, dysregulation of stemness signaling pathways in CSCs may enable CSCs to avoid chemotherapy induced cell death and also explain how the surviving CSCs may acquire the ability to metastasize to sites in the body that are distant from the primary tumor. Recent studies suggest the aberrant activity of CSCs stemness signaling pathways is reliant on the constitutive activation of the STAT3 transcription factor. STAT3 has therefore emerged as a promising target for inhibiting cancer stem cell survival and preventing metastasis.

Disclosed herein is a method for modulating STAT3 activity comprising binding one or more amino acid residues in STAT3's linker domain. In certain embodiments, STAT3 is bound to a STAT3 DNA binding site.

In certain embodiments, the binding to the linker domain comprises binding to at least one amino acid residue chosen from T515, D570, or K573 of SEQ ID NO: 1.

In certain embodiments, the binding to the linker domain comprises binding to T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, the method for modulating STAT3 activity comprises binding to one or more amino acid residues in a STAT3 DNA binding domain. In certain embodiments, the binding to the DNA binding domain comprises binding to at least one amino acid residue chosen from M331, H332, P333, D334, R335, P471, or M470 of SEQ ID NO: 1. In certain embodiments, the binding to the DNA binding domain comprises binding to M331, H332, P333, D334, R335, P471, and M470 of SEQ ID NO: 1. In certain embodiments, the binding reduces STAT3 binding affinity for a STAT3 DNA binding site.

Disclosed herein is also a method for modulating an activity of STAT3 comprising contacting one or more amino acid residues in STAT3's hinge pocket. In certain embodiments, STAT3 is bound to a STAT3 DNA binding site. In certain embodiments, the contact with the one or more amino acid residues in the hinge pocket comprises contacting one or more amino acid residues in STAT3's linker domain and DNA binding domain. In certain embodiments, the one or more amino acid residues in the DNA binding domain is chosen from M331, H332, P333, D334, R335, P471, or M470 of SEQ ID NO: 1. In certain embodiments, the one or more amino acid residues in the linker domain is chosen from T515, D570, or K573 of SEQ ID NO: 1. In certain embodiments, the contact with the one or more amino acid residues in the hinge pocket induces a change in STAT3 including increasing the distance between the SH2 domains of the STAT3 and/or bending the minor groove of the STAT3 DNA binding site.

Disclosed herein is also a method for modulating STAT3 activity in a cell comprising contacting one or more amino acid residues in STAT3's hinge pocket. The cell can be, for example, a cancer stem cell.

Disclosed herein is also a method for preventing or treating a disease or disorder associated with an aberrant STAT3 signaling pathway in a subject in need thereof comprising contacting one or more amino acid residues in STAT3's hinge pocket. In certain embodiments, the disease or disorder can be, for example, a cancer, an autoimmune disease, an inflammatory disease, inflammatory bowel diseases, arthritis, autoimmune demyelination disorder, Alzheimer's disease, stroke, ischemia reperfusion injury, or multiple sclerosis.

In certain embodiments, the cancer can be, for example, lung cancer, colon cancer, rectal cancer, colorectal cancer, gastrointestinal cancer, esophageal cancer, small bowel cancer, gastroesophageal junction cancer, appendiceal cancer, chondrosarcoma, adrenocorticoid cancer, laryngeal cancer, squamous cell carcinoma, angiosarcoma, leukemia, lymphoma, myeloma, brain cancer, ovarian cancer, melanoma, pancreatic cancer, gastric cancer, prostate cancer, breast cancer, liver cancer, renal cancer, myelodysplastic syndromes, cholangiocarcinoma, endometrial cancer, neuroendocrine cancer, bladder cancer, mesothelioma, synovial sarcoma, thymic cancer, Desmoid tumor, or head and neck cancer.

In certain embodiments, the cancer can be, for example, a metastatic cancer, a cancer that is refractory to chemotherapy, a cancer that is refractory to radiotherapy and/or a cancer that has relapsed.

Disclosed herein is a method for modulating an activity of STAT comprising inducing a conformational change in STAT3. In certain embodiments, the conformational change reduces STAT3's binding affinity for a STAT3 DNA binding site without altering the phosphorylation or dimerization of STAT3. In certain embodiments, the STAT3 can be, for example, bound to the STAT3 DNA binding site. In certain embodiments, the conformational change in the STAT3 increases the distance between the SH2 domains of STAT3. For example, the distance between the SH2 domains of the STAT3 can be increased by at least about 0.1 Ångström, at least about 0.2 Ångström, at least about 0.3 Ångström, at least about 0.4 Ångström, or at least about 0.5 Ångström. In certain embodiments, the method further comprises inducing a conformational change in the STAT3 DNA binding site bound by STAT3, for example, by creating a bend in the minor groove of the STAT3 DNA binding site. In certain embodiments, the bend in the minor groove of the STAT3 DNA binding site can be, for example, at least about 40 degrees. In certain embodiments, the conformational changes in STAT3 can reduce the binding affinity of STAT3 for the STAT3 DNA binding site by at least 20 fold. In certain embodiments, the conformational change in STAT3 can reduce the binding affinity of STAT3 for the STAT3 DNA binding site by at least about 200 fold. In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of one or more amino acid residues located within a hinge pocket of STAT3. For example, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least one of the following amino acid residues: M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1. In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least M331. In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least H332. In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least P333. In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least D334. In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least R335. In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least P471. In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least M470. In certain embodiments, the conformational change in STAT3 is induced by modifying the spatial configuration of at least T515. In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least D570. In certain embodiments, the conformational change in STAT3 is induced by modifying the spatial configuration of at least K573.

In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least two of the following amino acid residues: M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least three of the following amino acid residues: M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of at least four of the following amino acid residues: M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, the conformational change in STAT3 can be induced, for example, by modifying the spatial configuration of M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

Disclosed herein is also a method of modulating STAT3 activity in a cell comprising inducing a conformational change in cellular STAT3. In certain embodiments, the conformational change reduces the binding affinity of STAT3 for a STAT3 DNA binding site in a promoter of a STAT3 responsive gene. In certain embodiments, the cell can be, for example, a cancer stem cell. The STAT3 activity can be, for example, STAT3 binding to its cognate STAT3 DNA binding site or activation of a STAT3 responsive gene.

In certain embodiments, the disclosure provides a method of preventing or treating a disease or disorder associated with an aberrant STAT3 signaling pathway in a subject in need thereof comprising inducing a conformational change in cellular STAT3. In certain embodiments, the conformational change reduces the binding affinity of cellular STAT3 for a STAT3 DNA binding site in a promoter of a STAT3 responsive gene.

In certain embodiments, the disease or disorder comprises a cancer, an autoimmune disease, an inflammatory disease, inflammatory bowel diseases, arthritis, autoimmune demyelination disorder, Alzheimer's disease, stroke, ischemia reperfusion injury, or multiple sclerosis.

In certain embodiments, the cancer is lung cancer, colorectal cancer, gastrointestinal cancer, esophageal cancer, leukemia, lymphoma, myeloma, brain cancer, ovarian cancer, melanoma, pancreatic cancer, gastric cancer, prostate cancer, breast cancer, liver cancer, or head and neck cancer. In certain embodiments, the cancer is a metastatic cancer, a cancer that is refractory to chemotherapy or radiotherapy, a cancer that is resistant to chemotherapy or a cancer that has relapsed.

Disclosed herein are also methods of modulating an activity of a non-druggable protein comprising binding one or more amino acid residues in the protein's linker domain. In certain embodiments, the non-druggable protein comprises an effector domain and a binding domain, such as a nucleic acid binding domain. In certain embodiments, binding to the linker domain reduces the non-druggable protein's affinity for its cognate nucleic acid binding site.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows an exemplary embodiment of the inhibition of STAT3 by a Compound of the Disclosure in cultured mammalian cells. FIG.1A and Fig 1B show an exemplary STAT3-luciferase reporter expression in cells after activation by the addition of IL-6 (DLD1; FIG. 1A) or OSM (U87-MG; FIG. 1B) and cultured either in the presence or absence of a Compound of the Disclosure, e.g., 2-acetylnaphtho [2,3-b] furan-4,9-dione (also referred to herein as BBI-608). FIG. 1C shows an exemplary gel shift analysis of nuclear STAT3 from HeLa cells activated with OSM and treated with DMSO (control) or the indicated concentrations of the BBI-608. FIG. 1D shows an exemplary embodiment of a ChIP analysis of STAT3 occupancy on c-fos and myoglobin promoters in HeLa cells treated with or without BBI-608.

FIG. 2 shows an exemplary characterization of purified recombinant pSTAT3 and its interaction with a Compound of the Disclosure, e.g., 2-acetylnaphtho [2,3-b] furan-4,9-dione (also referred to herein as BBI-608). A Coomassie stained protein gel and corresponding Western blot of purified pSTAT3 produced in bacteria are shown in FIG. 2A and FIG. 2B respectively. FIG. 2C depicts an exemplary sedimentation velocity analysis that determines the dimerization status of the recombinant pSTAT3 at four different concentrations (2, 4, 8 and 16 µM). FIG. 2D shows an exemplary surface plasmon resonance (SPR) analysis of the interaction between the purified recombinant pSTAT3 protein, disclosed in Figs. 2A-C, and different concentrations of BBI-608. FIG. 2E shows an exemplary inhibition of recombinant pSTAT3 binding to its cognate DNA binding site by either a STAT3 inhibitor peptide (control) or the indicated concentrations of BBI-608. 

FIG.3 shows an exemplary embodiment of a Compound of the Disclosure, e.g., 2-acetylnaphtho [2,3-b] furan-4,9-dione (also referred to herein as BBI-608) bound to a pSTAT3/DNA complex. FIG. 3A depicts the overall structure of BBI-608 bound pSTAT3/DNA complex including the coiled-coil domain (CCD), the DNA binding domain (DBD), the linker domain (LD), the SH2 domain (SH2). The double strand DNA can be seen in the middle of the complex. The left monomer is shown by ribbon representation and the right monomer is shown by surface representation. The two BBI- 608 molecules and pY705 residues are shown by stick representation. The view is along the DNA axis. The Insert panel provides a close-up view of the hinge pocket bound to BBI-608. FIG. 3B illustrates the 2Fo-Fc map of the hinge pocket depicted in FIG. 3A (grey mesh at 1.5σ).

FIG.4 shows an exemplary embodiment of how a Compound of the Disclosure, e.g., 2-acetylnaphtho [2,3-b] furan-4,9-dione (also referred to herein as BBI-608) interacts with and inhibits the binding of pSTAT3 to its cognate DNA binding site. To visualize induced conformational changes within the pSTAT3/DNA complex, the BBI- 608 bound pSTAT3/DNA complex structure (shown in dark gray) was superimposed on the drug-free pSTAT3/DNA complex structure (shown in light gray; see EXAMPLE 4). FIG. 4A depicts a close-up view of the boxed area from FIG. 3A showing the induced pSTAT3 conformational changes. The arrows illustrate the direction of the domain shift induced by the binding of BBI-608 to the hinge pocket depicted in FIG. 3B. FIG. 4B shows the residues within the pSTAT3 hinge pocket that interact with BBI-608. FIG. 4C depicts an exemplary top view perspective of the superimposed structures. The arrows illustrate how the induced conformational change leads to the formation of ~ 0.5Å gap between the SH2 domains of STAT3. In contrast, monomeric STAT3 exhibits a more“closed hinge” conformation. FIG. 4D is an exemplary depiction of a 2Fo-Fc map of pY705’s phosphate group with residue R609 from another monomer (Gray mesh at 1.5σ).

FIG.5 shows an exemplary embodiment of a Compound of the Disclosure, e.g., 2-acetylnaphtho [2,3-b] furan-4,9-dione (also referred to herein as BBI-608), binding to a pSTAT3/DNA complex. FIG. 5A shows an exemplary embodiment of BBI-608, binding to the hinge pocket at the junction between the DNA binding domain (DBD) and the linker domain (LD) of the pSTAT3/DNA complex and inducing a conformational change that distorts the minor groove of the STAT3 DNA binding site (CCD: coiled-coil domain. SH2: SH2 domain). FIG. 5B shows the DNA duplex stretch and distortion induced by the binding of BBI-608, to STAT3. The image shows a bottom view of the pSTAT3/DNA complex bound to BBI-608, and superimposed on the drug-free pSTAT3/DNA complex structure but with all proteins hidden. The DNA bases and BBI-608 are shown by stick representation. The arrows to the left and right show the direction of the distortion in the DNA that is induced by the binding of Compound of the Disclosure to STAT3/DNA complex. An exemplary embodiment of the binding affinity of pSTAT3 for the STAT3 DNA binding site measured by isothermal titration calorimetry is shown either in the presence of DMSO (FIG. 5C) or BBI-608 (FIG.5D). FIG.5E shows an exemplary embodiment of the dissociation of p- STAT3/DNA complex from the BBI-608 mediated as measured by fluorescence polarization (FP). The black dots indicate the relative FP values (normalized to the values of free DNA). The curve represents the best fit using a one phase exponential decay model. The insert panel shows the same result plotted as percentage of inhibition. FIG. 5F shows an exemplary embodiment of the decrease in the binding affinity between a pSTAT3 dimer and its cognate DNA binding site induced by BBI-608. Double-stranded DNA plus DMSO or 2 μM BBI-608 was titrated with pSTAT3. The black dots and gray squares indicate the relative FP values (normalized to the values of free DNA) of pSTAT3/DNA binding with DMSO or with 2 μM BBI-608. The curves correspond to the fitted results using a one site binding model.

FIG. 6 demonstrates that a Compound of the Disclosure, e.g., 2-acetylnaphtho [2,3-b] furan-4,9-dione (also referred to herein as BBI-608) does not inhibit STAT1 or STAT5 DNA binding. FIG. 6A shows an exemplary embodiment of gel shift analysis of nuclear STAT1 from HeLa cells treated with either DMSO or the indicated concentrations of BBI-608 and stimulated with IFN-γ. FIG. 6B shows an exemplary embodiment of gel shift analysis of nuclear STAT5 from HeLa cells treated either with DMSO or the indicated concentrations of BBI-608 and stimulated with IFN-γ.

FIG. 7 shows a cartoon representation of the conformational changes in pSTAT3 induced by binding to a Compound of the Disclosure. The arrows indicate the direction of domain shifts.

FIG. 8 depicts the location of the STAT1 linker region mutations on the pSTAT3 structure in relation to the hinge pocket that binds to a Compound of the Disclosure (the mutations are converted into corresponding STAT3 residues).

The following are definitions of terms used in the present specification. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.

As used herein, the singular terms“a,”“an,” and“the” include the plural reference unless the context clearly indicates otherwise.

The phrase“and/or,” as used herein in the specification and in the claims, should be understood to mean“either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Thus, as a non-limiting example, a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example,“at least one of A and B” (or, equivalently,“at least one of A or B,” or, equivalently“at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

When the term“about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below those numerical values. In general, the term“about” is used herein to modify a numerical value above and below the stated value by a variance of 20%, 10%, 5%, or 1%. In certain embodiments, the term“about” is used to modify a numerical value above and below the stated value by a variance of 10%. In certain embodiments, the term“about” is used to modify a numerical value above and below the stated value by a variance of 5%. In certain embodiments, the term“about” is used to modify a numerical value above and below the stated value by a variance of 1%. When a range of values is listed herein, it is intended to encompass each value and sub-range within that range. For example,“1-5 mg” is intended to encompass 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 1-2 mg, 1-3 mg, 1-4 mg, 1-5 mg, 2-3 mg, 2-4 mg, 2-5 mg, 3-4 mg, 3-5 mg, and 4-5 mg.

In certain embodiments, a non-druggable protein refers to a protein of therapeutic interest that has, or had previously, proven inaccessible to high affinity binding by a drug, e.g. a small molecule or antigen-binding protein or antigen-binding fragment thereof. In certain embodiments, a non-druggable protein refers to a protein that has, or had previously, bound to a drug without altering protein function. In certain embodiments, a non-druggable protein refers to a protein whose change in function is, or is thought to be, without therapeutic benefit when the protein has, or had previously, bound to a drug,. In certain embodiments, a previously non-druggable protein can be a transcription factor. In certain embodiments, a previously non-druggable protein can be a transcription factor required for cancer stemness. In certain embodiments, a previously non-druggable protein can be a STAT transcription factor, e.g. STAT3.

In certain embodiments, STAT3 refers to mammalian STAT3. In certain embodiments, STAT3 refers to the human "Signal Transducer and Activator of Transcription 3" having the canonical 770 amino acid sequence of SEQ ID NO. 1 (Accession No: P40763-1; NP_644805.1):

Figure imgf000018_0001

STAT3, as used herein, includes pSTAT3 or pSTAT3 dimer. STAT3 α (770 aa, 92 kDa) has a highly conserved, modular structure containing multiple functional domains. The NH2-terminal domain (from about amino acid 1 to about amino acid 138 of SEQ ID No.: 1) is required for dimer–dimer interactions, cooperative DNA binding and nuclear translocation. The coiled-coil domain (from about amino acid 138 to about amino acid 321 of SEQ ID No.: 1) is essential for cytokine- and growth factor-stimulated recruitment of STAT3 to the receptor, dimer formation, nuclear translocation, and DNA binding. The DNA-binding domain (from about amino acid 321 to about amino acid 493 of SEQ ID No.: 1) recognizes the STAT3 DNA binding site as defined herein. The adjacent linker domain (from about amino acid 494 to about amino acid 583 of SEQ ID No.: 1) connects the DNA binding domain to the SH2 domain (from about amino acid 583 to about amino acid 688 of SEQ ID No.: 1) and facilitates docking of the protein to tyrosine phosphorylated receptor subunits and STAT3 dimerization. The STAT3 C-terminus (from about amino acid 688 to about amino acid 770 of SEQ ID No.: 1) contains an autonomously functioning transcriptional activation domain, absent from spliced isoforms of STAT1, STAT3, and STAT4 (reviewed in Lim and Cao Mol. BioSyst. (2006) 2, 536-550).

As used herein, the terms "linker domain" and "linker region" are used interchangeably to refer to the amino acid sequence situated between adjacent proteins domains of a target protein. In certain embodiments, the linker domain does not have a tertiary protein structure. In certain embodiments, the target protein is a previously non- druggable protein, e.g. a transcription factor. In certain embodiments, the target protein is a previously non-druggable protein, such as a nucleic acid binding transcription factor in which the linker region is situated between the transcription activation domain and the nucleic acid binding domain.

In certain embodiments, the term "linker domain" can refer to the linker domain of STAT3 corresponding to the amino acid residues from about amino acid 494 to about amino acid 583 of SEQ ID No.: 1. In certain embodiments, the STAT3 linker domain refers to a tertiary protein structure formed by a polypeptide comprising at least 10 contiguous amino acids of the amino acid sequence of SEQ ID NO.: 8:

Figure imgf000020_0001

In certain embodiments, the term "DNA binding domain" refers to the amino acid residues of STAT3 from about amino acid 321 to about amino acid 493 of SEQ ID No.: 1. In certain embodiments, the STAT3 DNA binding domain refers to a tertiary protein structure formed by a polypeptide comprising at least 10 contiguous amino acids of the amino acid sequence of SEQ ID NO.: 9:

Figure imgf000020_0002

As used herein, the term "STAT3 DNA binding site" refers to a double-stranded DNA sequence to which STAT3 can bind. In certain embodiments, the "STAT3 DNA binding site" can be, for example, a double-stranded DNA sequence comprising the sequence 5'-GATCCTTCTGGGAATTCCTAGATC-3' (SEQ ID NO.: 2) annealed to 3'- CTAGATCCTTAAGGGTCTTCCTAG-5' (SEQ ID NO.: 3). In certain embodiments, a DNA sequence that can be bound by STAT3 is referred to as STAT3's cognate DNA binding site. In certain embodiments, the "STAT3 DNA binding site" also refers to a consensus STAT3 DNA-recognition motif, called gamma-activated sites (GAS), in the promoter region of cytokine-inducible genes. Binding of STAT3 to these sites activates gene transcription.

As used herein, a "DNA binding activity of STAT3" may refer to the binding of STAT3 to its cognate DNA binding site, as defined herein. In the context of the instant disclosure, a STAT3-DNA complex refers to pSTAT3 homodimers binding to the STAT3 DNA binding site.

As used herein, the term "pSTAT3" refers to a STAT3 protein in which Tyr-705 is phosphorylated. "pSTAT3" comprises pSTAT3 dimers, e.g., pSTAT3 homodimers. "pSTAT3 phosphorylation" therefore refers to a pSTAT3 that is phosphorylated at Tyr- 705. pSTAT3 "dimerization" refers to the association of a pair of pSTAT3 molecules via binding of the SH2 domain of one STAT3 molecule to a phosphorylated Tyr-705 residue located on the other STAT3 molecule.

As used herein, the term "inducing a conformational change" refers to a modification in the three dimensional structure of a STAT3, including one bound to its cognate DNA binding site. In certain embodiments, the conformational change alters one or more functions of the STAT3 protein, including, but not limited to, the binding affinity of STAT3 for its cognate DNA binding site and/or STAT3 transcriptional activation.

In certain embodiments, the conformational change occurs within the STAT3 protein or a portion thereof. In certain embodiments, the "conformational change" comprises a shift in the position of one or more domains of STAT3, for example, the SH2 domains of STAT3. In certain embodiments, the "conformational change" comprises a movement of one or more amino acid residues or STAT3, for example, within the hinge pocket of STAT3.

In certain embodiments, the "conformational change" occurs within the STAT3 DNA binding site bound by STAT3. For example, the "conformational change" comprises a distortion, such as a bend, in the minor groove of the STAT3 DNA binding site bound by STAT3.

In certain embodiments, the term "inducing a conformational change" refers to those conformational changes in the STAT3-DNA complex that are induced by the binding of a Compound of the Disclosure to the hinge pocket of STAT3.

In certain embodiments, the term“a Compound of the Disclosure” refers to a molecule that contacts one or more amino acids in a linker region of a non-druggable protein. In certain embodiments, the term“a Compound of the Disclosure” refers to a molecule that contacts one or more amino acids in the linker region of STAT3 as defined herein. In certain embodiments, a Compound of the Disclosure refers to a molecule that binds non-covalently to the hinge pocket of STAT3, as defined herein. In certain embodiments, a Compound of the Disclosure refers to a molecule that binds covalently to the hinge pocket of STAT3, as defined herein.

In certain embodiments, a Compound of the Disclosure is not 2-acetylnaphtho [2,3-b] furan-4,9-dione (referred to herein as "BBI-608" or napabucasin).

In certain embodiments, the binding of a Compound to STAT3's hinge pocket reduces the binding affinity of STAT3 for its cognate DNA binding site by about 1-10 fold or about 10-50 fold or about 50-200 fold. In certain embodiments, a Compound of the Disclosure reduces the binding affinity of STAT3 for its cognate DNA binding site by at least 200 fold. In certain embodiments, a Compound of the Disclosure reduces the binding affinity of STAT3 for its cognate DNA binding site without altering STAT3 dimerization and/or STAT3 Tyr-705 phosphorylation.

In certain embodiments, a Compound of the Disclosure can bind to STAT3's hinge pocket, as defined herein, with a dissociation constant KD from about 10-6 M to about 10-12 M, from about 10-7 M to about 10-12 M, from about 10-8 M to about 10-12 M, from about 10-9 M to about 10-12 M, from about 10-10 M to about 10-12 M, from about 10- 11 M to about 10-12 M. In certain embodiments, a Compound of the Disclosure can bind to STAT3's hinge pocket, as defined herein, with a dissociation constant KD from about 10-6 M to about 10-11 M, from about 10-6 M to about 10-10 M, from about 10-6 M to about 10-9 M, from about 10-6 M to about 10-8 M, or from about 10-6 M to about 10-7 M. In certain embodiments, a Compound of the Disclosure can bind to STAT3 with a KD from about 10-7 M to about 510-7 M. In certain embodiments, a Compound of the Disclosure can bind to STAT3 with a KD from about 10-7 M to about 210-7 M.

In certain embodiments, a Compound of the Disclosure can be, for example, a synthetic organic molecule. In certain embodiments, the Compound of the Disclosure can be, for example, a small molecule. In certain embodiments, a Compound of the Disclosure can be, for example, a small molecule having a molecular weight from about 100 to about 10000 Daltons, from about 100 to about 5000 Daltons, from about 100 to about 1000 Daltons or from about 100 to about 500 Daltons. In certain embodiments, a Compound of the Disclosure can be, for example, a small molecule having a size from about 0.01 nm to about 5 nm, from about 0.01 to about 1nm, from about 0.01 to about 0.5nm, from about 0.01 to about 0.1 nm, or about 0.01 to about 0.05 nm. In certain embodiments, the small molecule can be planar. In certain embodiments, the small molecule comprises a ring structure, for example, a naphthoquinone ring structure. In certain embodiments, the Compound of the Disclosure is 2-acetylnaphtho [2,3-b] furan- 4,9-dione, also referred to herein as "BBI-608" or napabucasin.

In certain embodiments, a Compound of the Disclosure refers to a molecule, for example, a peptide, polysaccharide or nucleic acid, e.g., an oligonucleotide aptamer, which can contact at least one amino acid within STAT3's hinge pocket as defined herein.

In certain embodiments, a Compound of the Disclosure refers to a molecule, for example, a peptide, polysaccharide or nucleic acid, e.g., an oligonucleotide aptamer, which can contact at least one amino acid selected from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure can be, for example, a synthetic antibody mimic (SyAM) (McEnaney et al., J Am Chem Soc. 2014 Dec 31; 136(52):18034-43).

In certain embodiments, a Compound of the Disclosure can be, for example, a biological molecule that can contact at least one amino acid within STAT3's hinge pocket. In certain embodiments, a Compound of the Disclosure can be, for example, an antigen-binding protein, or antigen-binding fragment thereof, that can specifically bind to STAT3's hinge pocket as defined herein. In certain embodiments, a Compound of the Disclosure can be, for example, an antigen-binding protein, or antigen-binding fragment thereof, that can bind to at least one amino acid selected from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least one amino acid, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten amino acids within STAT3's hinge pocket as defined herein.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least one amino acid, at least two or at least three amino acids chosen from the amino acid sequences of SEQ ID NO: 8 or 9.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least one amino acid, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten amino acids chosen from the amino acid sequence of SEQ ID NO: 10.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least one amino acid chosen from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least two amino acids chosen from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least three amino acids chosen from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least four amino acids chosen from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1. In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least five amino acids chosen from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least six amino acids chosen from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least seven amino acids chosen from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least eight amino acids chosen from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact at least nine amino acids chosen from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, a Compound of the Disclosure refers to a molecule that can contact M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

As used herein, the term“an antigen-binding protein” can refer to naturally occurring or man-made antibodies such as monoclonal antibodies produced by conventional hybridoma technology. The term "antigen-binding protein" can refer to monoclonal and polyclonal antibodies as well as fragments containing the antigen- binding domain and/or one or more complementarity determining regions of these antibodies that can specifically bind to the hinge pocket of STAT3 as defined herein. Thus, in certain embodiments, the term“antigen-binding protein” encompasses a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule that in combination form a specific binding site that can contact at least one amino acid residue within STAT3's hinge pocket as defined herein. In certain embodiments, the antigen- binding protein is an IgG antibody or antigen-binding fragment thereof. For example, the antigen-binding protein can be an IgG1, IgG2, IgG3, or IgG4 antibody or antigen- binding fragment thereof. In certain embodiments, the antigen-binding protein, or antigen-binding fragment thereof, can be modified, for example, by conjugation or glycosylation, such as sialylation or galactosylation.

In certain embodiments, the term "antigen-binding protein" also encompasses a recombinant antibody, or antigen-binding fragment thereof, that is generated in cell culture, in phage, or in various animals, including, but not limited to, cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes. In certain embodiments, a Compound of the Disclosure can be, for example, a recombinant antibody including, but are not limited to, CrossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, Nanobody, Nanobody-HSA, BiTE, Diabody, DART, Knobs-in-holes common LC, Knobs-in-holes assembly, Charge pair, Fab-arm exchange, SEEDbody, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Triomab, LUZ-Y, Fcab, κλ−body, Orthogonal Fab, Miniantibody, Minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv- KIH, Fab- scFv-Fc, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, Dock and Lock, ImmTAC, HSAbody, scDiabody-HSA, Tandem scFv- Toxin, DVI-IgG (four-in-one), Cov-X-Body, IgG-IgG and scFv1-PEG- scFv2 or an antigen-binding fragment thereof (reviewed in Spiess et al. Mol Immunol. (2015); 67(2 Pt A):95-106).

For example, the Compound of the Disclosure can be a multi-specific recombinant antibody, or antigen-binding fragment thereof, that binds to an epitope within the hinge pocket of STAT3 and an epitope of another antigen of therapeutic interest, for example, a checkpoint inhibitor. In another example, a Compound of the Disclosure can be a multi-specific recombinant antibody, or antigen-binding fragment thereof, that binds to an epitope within the hinge pocket of STAT3 and another epitope either on the same STAT3 protein molecule or on a different STAT3 protein molecule. In another example, a Compound of the Disclosure can be a multi-specific recombinant antibody, or antigen-binding fragment thereof, that binds to an epitope within the hinge pocket of STAT3 and phosph-Tyr705 either on the same STAT3 protein molecule or on a different STAT3 protein molecule.

In certain embodiments, binding of a Compound of the Disclosure to phosph- Tyr705 disrupts phosph-Tyr705 - SH2 interaction and STAT3 dimerization.

Examples of a recombinant antibody, or antigen-binding fragment thereof, that can bind to STAT3's hinge pocket includes, but is not limited to, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 1990/05144, the content of which is incorporated herein by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term“antigen-binding protein." Other forms of single chain antibodies, such as diabodies are also encompassed within the term “antigen-binding protein." Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-54041354-5). Single chain antibodies can also include“linear antibodies” comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870). Dual Variable domain immunoglobulins (DVDs; see U.S. Patent No. 7,612,181, the content of which is incorporated herein in its entirety) are also encompassed within the term "antigen- binding protein." Methods of generating highly diverse libraries of recombinant antigen- binding molecules and screening them for binding to a targeted epitope, e.g. STAT3's hinge pocket, are well known in the art (see, for example, Hoogenboom, Nature Biotechnology (2005) 23, 1105 - 1116).

In certain embodiments, the Compound of the Disclosure can be, for example, a STAT3-specific single domain antibody (STAT3-specific sdAb or STAT3-specific VHH) that contacts one or more amino acid residues within STAT3's hinge pocket, as defined herein. Methods of making sdAb targeting STAT3 are disclosed in PCT/US2015/057223, the content of which is incorporated by reference herein in its entirety. In certain embodiments, a“single domain antibody,”“sdAb” or“VHH” refers to a polypeptide or protein comprising an amino acid sequence having four framework regions interrupted by three complementarity determining regions (FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4). In certain embodiments, a sdAb also includes a polypeptide or protein having a sdAb amino acid sequence. sdAbs can be produced in camelids such as llamas, but can also be synthetically generated using recombinant techniques that are well known in the art. In certain embodiments, sdAbs can be humanized. In certain embodiments, sdAbs can be subjected to molecular evolution and high affinity binders can be selected using methods that are well known in the art (see, for example, Published U.S. Patent Application No. 2015/0158934, the content of which is incorporated by reference herein in its entirety).

In certain embodiments, a Compound of the Disclosure can be, for example, a STAT3 binding molecule that binds outside of the hinge pocket and induces a conformational change in STAT3 and/or the STAT3 DNA binding site, as described herein. In certain embodiments, a Compound of the Disclosure can be, for example, a STAT3-specific antigen-binding protein that binds outside of the hinge pocket and induces a conformational change in STAT3 and/or the STAT3 DNA binding site, as described herein. In certain embodiments, a Compound of the Disclosure can be, for example, a STAT3-specific binding molecule that induces a conformational change in STAT3 and/or the STAT3 DNA binding site, as described herein, as well as reducing the binding affinity of STAT3 for its cognate DNA binding site by about 1-10 fold or about 10-50 fold or about 50-200 fold.

Interaction of a Compound of the Disclosure with a linker domain of a target protein can be measured using methodologies that are well known in the art including, but are not limited to, affinity-based techniques (e.g., Nuclear magnetic resonance (NMR)-based screening, affinity selection mass spectrometry (AS-MS), Surface plasmon resonance (SPR)), and stability based techniques (e.g. Differential scanning fluorimetry (DSF), Hydrogen-deuterium exchange (HDX) coupled with NMR or mass spectrometry (reviewed by Makley et al. Chem Biol Drug Des.2013 Jan; 81(1): 22–32). For example, surface plasmon resonance (SPR) technology provides a rapid and cost effective means of determining the affinity, specificity, and kinetics of biomolecular interactions in real time making it an ideal tool for use in high throughput applications.

In certain embodiments, a Compound of the Disclosure refers to a molecule that competes with another molecule for binding to one or more amino acid residues within STAT3's hinge pocket as defined herein. In certain embodiments, a Compound of the Disclosure can be, for example, a molecule that competes with an antibody for binding to STAT3's hinge pocket as defined herein. In certain embodiments, a Compound of the Disclosure can be, for example, a molecule that competes with an antibody for binding to at least one amino acid residue within STAT3's hinge pocket as defined herein. In certain embodiments, a Compound of the Disclosure can be, for example, a molecule that competes with a small molecule for binding to STAT3's hinge pocket as defined herein. In certain embodiments, a Compound of the Disclosure can be, for example, a molecule that competes with 2-acetylnaphtho [2,3-b] furan-4,9-dione (referred to herein as "BBI-608." or napabucasin) for binding to STAT3's hinge pocket as defined herein.

As used herein, the "hinge pocket" refers to a region located between the DNA binding domain and the linker domain of STAT3. In certain embodiments, the hinge pocket of STAT3 refers to the amino acid sequence from about amino acid 321 to about amino acid 583 of SEQ ID No.: 1. In certain embodiments, the hinge pocket of STAT3 domain refers to a tertiary protein structure formed by a polypeptide comprising at least 10 contiguous amino acids of the amino acid sequence of SEQ ID NO.: 10:

Figure imgf000032_0001

In certain embodiments, the "hinge pocket" of STAT3 refers to the hinge pocket of STAT3 α or STAT3 β. In certain embodiments, the term "contacting" or "binding" means forming a noncovalent bond with one or more amino acids of the hinge pocket of STAT3 as defined herein. In certain embodiments, the one or more amino acids of the hinge pocket of STAT3 include at least one amino acid selected from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

In certain embodiments, the term "contacting" or "binding" can refer to forming a covalent bond with one or more amino acids of the hinge pocket of STAT3 as defined herein. In certain embodiments, the one or more amino acids of the hinge pocket of STAT3 include at least one amino acid selected from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

As used herein, the phrase "modifying the spatial configuration" refers to the changes in the position and/or orientation of one or more amino acids in the hinge pocket of STAT3. In certain embodiments, the change results in a conformational change in the STAT3-DNA complex as disclosed herein. In certain embodiments, binding of a Compound of the Disclosure to the STAT3 hinge pocket modifies the spatial configuration of at least one amino acid selected from M331, H332, P333, D334, R335, P471, M470, T515, D570, and K573 of SEQ ID NO: 1.

As used herein, a "STAT3 responsive gene" refers to a gene having a functional STAT3 DNA binding site in its promoter that, when bound by STAT3, activates transcription of the gene. Examples of STAT3 regulated genes can be found, for example, in Dauer et al. (2005) Oncogene 24(21): 3397-3408.

As used herein, an "aberrant STAT3 signaling pathway" refers to signaling pathways that activate STAT3 constitutively. The term“subject” generally refers to a mammal or mammalian cell, including a human or human cell. The term also refers to an organism, which includes a cell or a donor or recipient of such cell. In certain embodiments, the term“subject” refers to any animal including, but not limited to humans, mammals and non-mammals, such as non- human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, fish, nematode, and insects. Under some circumstances, the terms“subject” and “patient” are used interchangeably herein in reference to a human subject.

As used herein, the terms“treatment,” or“treating” are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and/or prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. In certain embodiments, treating a subject refers to inducing a conformational change in cellular STAT3, for example, that reduces the affinity of STAT3 for its cognate DNA binding site and subsequently the expression of STAT3 responsive genes, e.g. genes required for cancer stemness.

The term“cancer” in a subject refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain morphological features. Often, cancer cells can be in the form of a tumor or mass, but such cells may exist alone within a subject, or may circulate in the blood stream as independent cells, such as leukemic or lymphoma cells. Examples of cancer, as used herein, include, but are not limited to, lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, breast cancer, uterine cancer, ovarian cancer, peritoneal cancer, colon cancer, rectal cancer, colorectal adenocarcinoma, cancer of the anal region, stomach cancer, gastric cancer, gastrointestinal cancer, gastric adenocarcinoma, adrenocorticoid carcinoma, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, chondrosarcoma, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, Ewing’s sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, bladder cancer, testicular cancer, cancer of the ureter, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, kidney cancer, renal cell carcinoma, chronic or acute leukemia, lymphocytic lymphomas, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Some of the exemplified cancers are included in general terms and are included in this term. For example, urological cancer, a general term, can include bladder cancer, prostate cancer, kidney cancer, testicular cancer, and the like; and hepatobiliary cancer, another general term, includes liver cancers (itself a general term that includes hepatocellular carcinoma or cholangiocarcinoma), gallbladder cancer, biliary cancer, or pancreatic cancer. Both urological cancer and hepatobiliary cancer are contemplated by the present disclosure and included in the term“cancer.” As used herein,“cancer” can include the term,“solid tumor.” As used herein, the term“solid tumor” refers to those conditions, such as cancer, that form an abnormal tumor mass, such as sarcomas, carcinomas, and lymphomas. Examples of solid tumors include, but are not limited to, non-small cell lung cancer (NSCLC), neuroendocrine tumors, thyomas, fibrous tumors, metastatic colorectal cancer (mCRC), and the like. In certain embodiments, the solid tumor disease can be, for example, an adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and the like.

In certain embodiments, the cancer can be, for example, esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, gastric cancer, chondrosarcoma, colorectal adenocarcinoma, breast cancer, ovarian cancer, head and neck cancer, melanoma, gastric adenocarcinoma, lung cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, cervical cancer, brain tumor, multiple myeloma, leukemia, lymphoma, prostate cancer, cholangiocarcinoma, endometrial cancer, small bowel adenocarcinoma, uterine sarcoma, or adrenocorticoid carcinoma. In certain embodiments, the cancer can be, for example, esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, colorectal adenocarcinoma, breast cancer, ovarian cancer, head and neck cancer, melanoma, gastric adenocarcinoma, lung cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, cervical cancer, brain tumor, multiple myeloma, leukemia, lymphoma, prostate cancer, cholangiocarcinoma, endometrial cancer, small bowel adenocarcinoma, uterine sarcoma, or adrenocorticoid carcinoma. In certain embodiments, the cancer can be, for example, breast cancer. In certain embodiments, the cancer can be, for example, colorectal adenocarcinoma. In certain embodiments, the cancer can be, for example, small bowel adenocarcinoma. In certain embodiments, the cancer can be, for example, hepatocellular carcinoma. In certain embodiments, the cancer can be, for example, head and neck cancer. In certain embodiments, the cancer can be, for example, renal cell carcinoma. In certain embodiments, the cancer can be, for example, ovarian cancer. In certain embodiments, the cancer can be, for example, prostate cancer. In certain embodiments, the cancer can be, for example, lung cancer. In certain embodiments, the cancer can be, for example, uterine sarcoma. In certain embodiments, the cancer can be, for example, esophageal cancer. In certain embodiments, the cancer can be, for example, endometrial cancer. In certain embodiments, the cancer can be, for example, cholangiocarcinoma. In certain embodiments, the aforementioned cancers can be, for example, unresectable, advanced, refractory, recurrent, or metastatic.

Provided herein is a Compound of the Disclosure that binds to and inhibits STAT3 in vitro and in vivo (see EXAMPLES 1 and 2). Surprisingly, co-crystallization of a Compound of the Disclosure bound within a complex of a phosphorylated STAT3 dimer itself bound to a STAT3 DNA binding site (see EXAMPLE 3) revealed that the Compound binds to a pocket within the hinge domain of STAT3 that is distant from the SH2 domain dimerization interface and was also outside of the DNA binding domain.

The conformational changes resulting from the binding of a Compound of the Disclosure to the hinge pocket were deduced by superimposing the structure of the STAT3-DNA complex bound by the Compound on the structure of the previously published drug-free STAT3/DNA complex structure (Becker et al. Nature (1998) 394, 145-151; PDB id: 1BG1) (see EXAMPLE 4 and FIG.4).

As shown in FIG. 4A, the hinge pocket residues D334, R335, T515, D570 and K573 re-orientated their side chains upon binding to a Compound of the Disclosure. Binding of the Compound induced a conformational change in the DNA binding domain and a significant change in the linker domain. Notably, the C-terminal ends of the α-helices α6 and α8 of the linker domain were shifted towards the DNA binding domain. The nearby short α-helix in the SH2 domain, previously named DB, was also involved in this conformational shift. Although it is not intended that the present disclosure be limited to any particular theory or hypothesis, these alterations suggest that the local conformational changes induced by binding a Compound of the Disclosure were propagated from the linker domain to the SH2 domain, causing the entire upper region of STAT3 to shift towards the lower domains. When the Compound-bound STAT3 and the drug-free STAT3 structures were superimposed by their lower domains, the average structural deviation between the two domains was about 0.3 Å for all 350 Cα atoms; however, the structural deviation between the STAT3 upper arms was more than doubled to about 0.7 Å for all 210 Cα atoms.

Interestingly when DNA was absent, as shown in the previously published monomeric STAT3 core structure without DNA (Ren et al. (2008) 374, 1-5; PDB id: 3CWG), the STAT3 hinge pocket adopted a“closed” conformation (FIG. 4B) which suggests that STAT3 protein must open up from its hinge domain and reorient itself into an“open” conformation in order to accommodate the DNA molecule, and also extend its upper domains to form the dimer contact over the DNA. In order to accommodate its target DNA, the STAT3 monomers assumed an“open” configuration and extended their SH2 domains forming a tight dimer between the two STAT3 molecules via the pTyr705-SH2 interaction. Although it is not intended that the present disclosure be limited to any particular theory or hypothesis, it is suggested that the Compound seemed to act as a“double-sided tape” that tightened the hinge domain via multiple strong interactions with both the upper and lower domains (see TABLE 3 in EXAMPLE 3), locking STAT3 in an “intermediate” state between the “closed” and “open” conformations. Although it is not intended that the present disclosure be limited to any particular theory or hypothesis, it is also suggested that upon binding of the Compound, the STAT3 SH2 domains shifted away from each other forming a relatively loose pTyr705-SH2 dimer over the target DNA. Without intended to be limited by any particular theory or hypothesis, it is also suggested that this shift created a ~0.5Å gap between the STAT3 monomers in the dimer (see FIG.4C). The 2Fo-Fc density map also clearly showed a more distanced backbone trace of the pY705-SH2 dimerization segments that did not overlap with the same fragments in the STAT3 structure (FIG.4C and 4D). The ~0.5Å gap between STAT3 induced by binding the Compound further destabilized the STAT3/DNA complex. A cartoon representation illustrating the conformational changes at the linker domain and SH2 domain induced by binding a Compound of the Disclosure is presented in FIG.7.

Although it is not intended that the present disclosure be limited to any particular theory or hypothesis, it is suggested that in addition to inducing STAT3 conformational changes, binding the Compound also triggered secondary conformational changes in DNA. The target STAT3 DNA binding site that contains the STAT3 binding GAS consensus motif adopted a B-form conformation with a 40° bend at the minor groove in the middle of the bound DNA region (FIG 5A). Although it is not intended that the present disclosure be limited to any particular theory or hypothesis, it is also suggested that the Compound appeared to be in close contact with the phosphate group of the nucleotide on consensus GAS position +1 (FIG.3B). The distance between the outside ketone oxygen of the naphthoquinone and the phosphate oxygen atoms of nucleotide G11 was 3.8 Å (see FIG. 3B). By virtue of the repulsion between the two oxygen atoms, the Compound slightly pushed the DNA molecule away from STAT3 (FIG. 5B). Although it is not intended that the present disclosure be limited to any particular theory or hypothesis, it is also suggested the Compound-bound STAT3 monomer pushed the eight nucleotides between position +1 and position +8 of the GAS DNA consensus motif toward their 3’ direction by about 0.5 Å (FIG. 5B). The base of this nucleotide was also re-orientated to pair with the unshifted nucleotide located on the opposite strand, this in turn increased the distance between the base pairs and created a distortion within each DNA strand (FIG. 5B). Thus, although it is not intended that the present disclosure be limited to any particular theory or hypothesis, it is also suggested that binding to the hinge pocket in STAT3 triggers conformational changes and locks STAT3 into an “intermediate” configuration between the “closed” and “open” conformations, resulting in reduced affinity for DNA.

Although it is not intended that the present disclosure be limited to any particular theory or hypothesis, it is also suggested that in analogy to allosteric inhibitors, the binding of the Compound to STAT3, at a site distant from STAT3's DNA binding domain, induced inhibitory conformational changes that led to dramatically reduced affinity between STAT3 and its target DNA, resulting in direct inhibition of STAT3 transcription function. Targeting of the allosteric-like pocket in the hinge domain of STAT3 for drug development is consistent with reports about a linker domain mutant of STAT1 (Yang et al. (2002) The Journal of Biological Chemistry 277, 13455-13462; Yang et al. Mol. Cell. Biol. (1999) 19, 5106-5112) and the protein dynamic allostery hypothesis (Ma et al. Structure (2011) 19, 907-917). In comparison with its wild-type counterpart, this STAT1 mutant (K554A, E555A) exhibits a substantial reduction in STAT1 residence time on DNA in vitro explaining the decreased transcriptional activity of the mutant. This linker domain mutation appears to break down a strong“double salt-bridge” interaction with the DNA binding domain (FIG. 8). Loosening of these strong ties between the linker domain and DNA binding domain might greatly increase the intrinsic flexibility of STAT1 protein, affecting its DNA binding properties. Although it is not intended that the present disclosure be limited to any particular theory or hypothesis, it is also suggested that in the structure of STAT3, the Compound bound to a hinge pocket that is in close proximity to this mutation site (FIG. 8). However, although it is not intended that the present disclosure be limited to any particular theory or hypothesis, it is also suggested that, instead of loosening the interactions between the linker domain and DNA binding domain, the Compound induced conformational changes and decreased the binding of STAT3 to DNA. These findings suggest the hinge domain may have an important function in regulating DNA-binding activity in addition to its known structural role. EXAMPLES

Examples are provided below to further illustrate different features of the present disclosure. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention. EXAMPLE 1: A Compound of the Disclosure inhibits STAT3-driven transcription and

STAT3 binding to its canonical DNA binding site in vivo Inhibition of STAT3-driven transcription in DLD1 and U87-MG cells To determine the in vivo biological activity and specificity of a Compound of the Disclosure, 2-acetylnaphtho [2,3-b] furan-4,9-dione (referred to herein as BBI-608), on STAT3 function, DLD1 cells (ATCC No.: CCL-221; a human Dukes' type C, colorectal adenocarcinoma cell line) and U87-MG cells (ATCC No.: HTB1; a human glioblastoma/astrocytoma cell line) were first stably transfected with a STAT3- dependent Photinus pyralis (firefly) luciferase reporter (Affymetrix; cat #: LR0077). A Renilla luciferase reporter having only a minimal promoter acted as a control. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gemini) and 1% penicillin/streptomycin (Life Technologies). Cells were treated for 30 min with the indicated concentration of the BBI-608 (shown along x axis: 0.157 ^ ^M; 0.3125 ^ ^M; 0.625 ^ ^M; 1.25 ^ ^M; 2.5 ^ ^M; 5 ^ ^M; and 10 ^M) followed by stimulation with 10 ng/ml Oncostatin-M (U87-MG cells) or 20 ng/ml IL-6 (DLD1 cells) for 4 hours. IL-6 and Oncostatin-M were purchased from R&D Systems. Cells were then harvested and the levels of Photinus pyralis (firefly; FIG.1A, dark gray bar graph) and Renilla luciferase (FIG.1A, light gray bar graph) were determined using a Dual-Glo luciferase assay system (Promega; cat #: E2920) according to the manufacturer's instructions.

Treatment of DLD-1 cells with IL-6 resulted in a significant induction of STAT3-dependent firefly luciferase reporter activity. As shown in FIG. 1A, concentrations of the BBI-608 at or above 1.25 μM inhibited the STAT3 transcriptional activation by more than 95%. In contrast, the BBI-608 had no detectable effect on the activity of the control Renilla luciferase reporter under similar conditions (see FIG.1A, light gray bar graph). Similarly, STAT3 reporter activity after OSM treatment in U87- MG cells was inhibited by about 90% at concentrations of the BBI-608 between 0.625 and 1.25 μM (FIG.1B).

Inhibition of STAT3 DNA binding activity in vivo

STAT3 DNA binding activity in cells was tested in the presence or absence of the BBI-608. Hela cells (ATCC No.: CCL2) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gemini) and 1% penicillin/streptomycin (Life Technologies). Non-confluent HeLa cells were treated for 2 hours with DMSO or 100nM, 300nM or 600nM of the BBI-608 followed by stimulation with 10 ng/ml Oncostatin-M (R&D Systems) for 15 min. The cells were then harvested and nuclear extracts were prepared according to manufacturer’s instructions (EMD Millipore).

A 24 bp probe comprising a STAT3 DNA binding site was generated by annealing the single-stranded oligonucleotide 5'- GATCCTTCTGGGAATTCCTAGATC-3' (SEQ ID NO.: 2) to its complementary sequence (purchased from Santa Cruz, cat #: sc-2571). The probe was 32 ^P end-labeled using T4 polynucleotide kinase (Perkin Elmer).10 ^g of nuclear extract and the labelled probe were then incubated at room temperature for 30 min in 1x gel shift binding buffer (Promega, cat #: E358A (5 X); 1 X gel shift binding buffer: 4% glycerol; 10 mM Tris- HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.5 mM EDTA and 50 ng of poly (dI-dC)). Following incubation, the mixtures were separated on a native 4% polyacrylamide gel (PAGE), and bands were visualized by autoradiography. For purified protein, 50 ng of STAT3 was incubated with DMSO, the indicated concentrations of BBI-608, or with STAT3 inhibitor peptide (EMD Millipore) for 15 min at room temperature.

As can be seen in FIG. 1C, the binding of STAT3 to its cognate DNA binding site was reduced by about 5-10 fold by 600 nM of the BBI-608.

Inhibition of DNA binding is specific to STAT3

Hela cells (ATCC No.: CCL2) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gemini) and 1% penicillin/streptomycin (Life Technologies). Non-confluent HeLa cells were treated for 2 hours with DMSO or 100nM, 300nM or 600nM of the BBI-608 followed by stimulation with 5 ng/ml IFN- ^ ^for 1 ^ ^min prior to harvesting. Nuclear extracts were prepared according to manufacturer’s instructions (EMD Millipore). Gel shift analysis of nuclear STAT1 or STAT5 was then performed as described in Example 1 using oligonucleotides containing either a STAT1 or STAT5 DNA binding site.

FIG. 6 shows that BBI-608 had no significant effect on the formation of complexes (indicated by arrows) comprising either pSTAT1 bound to the STAT1 binding site (FIG. 6A) or pSTAT5 bound to the STAT5 binding site (FIG. 6B). The inhibition of DNA binding by Compound of the Disclosure therefore appears to be specific to STAT3.

Inhibition of STAT3 DNA binding activity at an endogenous STAT3 responsive promoter

Binding of STAT3 to the endogenous STAT3 responsive c-fos promoter was determined by chromatin immunoprecipitation (ChIP) using an iDeal ChIP kit for transcription factors as described by the manufacturer (Diagenode). Cross-linked chromatin from 4 x 106 HeLa cells was treated for 2 hours with either DMSO or 1 µM BBI-608 followed by incubation with either 5 μl of STAT3α (D1A5) XP Rabbit mAb (Cell Signaling Technology, cat#: 8768) or 2 μl of Normal Rabbit IgG (Cell Signaling) using DiaMag Protein A-coated magnetic beads. The enriched DNA was analyzed by real-time PCR using c-fos promoter (Cell Signaling) or myoglobin promoter primers (Diagenode). The amount of immunoprecipitated DNA in each sample relative to the total amount of input chromatin (equivalent to 1) was then calculated.

As shown in FIG.1D, 1 μM of BBI-608 inhibited chromosomal binding at the c- fos promoter by about 70%. No STAT3 binding to the control myoglobin gene was detected. No inhibition of STAT3 signaling pathway kinases

To exclude the possibility that the BBI-608 inhibited STAT3 activity in cells through direct kinase inhibition, the in vitro activity of an array of oncology-related kinases implicated in the STAT3 signaling pathway, such as Janus kinases and Src family kinases, was tested by Eurofins in the presence of a high dose (30 ^M) of the BBI-608. As can be seen in TABLE I below, the BBI-608 failed to inhibit any of the kinases tested in vitro. These data therefore suggest that the BBI-608 acted directly on STAT3 to inhibit cytokine-dependent STAT3 transcriptional activity in vivo.

Figure imgf000045_0001
EXAMPLE 2: A Compound of the Disclosure binds directly to STAT3 Protein expression and purification of recombinant STAT3

To determine if a Compound of the Disclosure, 2-acetylnaphtho [2,3-b] furan- 4,9-dione (also referred to herein as BBI-608), binds directly to STAT3 in vitro, recombinant phosphorylated STAT3 (pSTAT3) was expressed in and purified from E. coli (FIG.2). DNA sequence of human STAT3 corresponding to murine STAT3 protein fragment (127-722) was cloned into a pET32a vector. STAT3 was expressed and tyrosine phosphorylated in TKB1 cells according to manufacturer's instructions (Agilent Technologies). The BL21(DE3) TK strain, abbreviated TKB1 (a tyrosine kinase (TK) derivative of the BL21 (DE3) strain (E. coli B F dcm ompT hsdS(rBmB) gal λ(DE3) [pTK Tetr]; Agilent Technologies, cat#: 200134), carries the gene for T7 RNA polymerase in the chromosome under the control of the lacUV5 promoter. Induction of the polymerase with isopropyl-β-D-thio-galactopyranoside (IPTG) allows controlled protein expression of the STAT3 coding sequence placed downstream of the T7 RNA polymerase binding site. The TKB1 strain also harbors a plasmid-encoded, inducible tyrosine kinase gene (pTK) that drives the phosphorylation of the STAT3 Tyr705 residue (pSTAT3) which is required for the activation of STAT3 that triggers STAT3 dimerization, nuclear translocation, and DNA binding in vivo.

pSTAT3 was purified from TKB1 cells based on a modified protocol previously published by Becker (Becker et al. (1998) Expression of a tyrosine phosphorylated, DNA binding STAT3 beta dimer in bacteria. FEBS letters 441, 141-147). Following the induction of STAT3 protein expression and phosphorylation, the TKB1 cells were centrifuged and the cell pellet was re-suspended in lysis buffer (20 mM HEPES pH 7.0, 0.1 M KCl, 10% glycerol, 1 mM EDTA, 10 mM MgCl2, and 5 mM DTT) and the cells were disrupted in a microfluidizer. The lysate was pelleted by centrifugation for 40 min at 40,000 g at 4ºC. 0.1% polyethyleneimine (PEI) was added to the stirred supernatant for 15 min at 4ºC. An additional 20 minute centrifugation at 30,000 g was performed to remove nucleic acids. Ammonium sulfate was added slowly to the solution up to 35% saturation at 4ºC with stirring. The precipitated pSTAT3 was collected by a 20 min centrifugation at 40,000 g, resuspended in a SEC running buffer (20 mM HEPES pH 7.0, 200 mM NaCl, 10 mM MgCl2, 5 mM DTT) and dialyzed overnight at 4ºC against 4L SEC running buffer. The filtered protein solution was then loaded on a HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated with the SEC running buffer and a single major peak was eluted from the column.

SDS PAGE and Western blot analysis

The eluted pSTAT3 protein was analyzed by SDS PAGE to test for homogeneity (FIG.2A) and Western blot (FIG.2B) to confirm phosphorylation of Tyr705 of STAT3.

TKB1 cells expressing pSTAT3 were washed twice with ice-cold phosphate- buffered saline (PBS) and lysed in lysis buffer (50 mM HEPES, pH 7.5, 1% Nonidet P- 40, 150 mM NaCl, 1 mM EDTA, 1X protease inhibitor cocktail (EMD Millipore)). 20 ^g of soluble protein was separated by SDS-PAGE and Coomassie stained followed by transfer to PVDF membranes. pSTAT3 was then detected using a p705-STAT3- specific (Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb, cat #9145) primary antibody purchased from Cell Signaling Technology. The antigen-antibody complexes were visualized by enhanced chemiluminescence (BioRad).

As can be seen in Fig. 2A, SDS PAGE analysis of the purified STAT3 revealed a single species of protein on Coomassie staining of the SDS-PAGE (see FIG.2A) that co-migrated with pSTAT3 detected by Western blot analysis of the SDS-PAGE gel (see FIG.2B).

Analytical ultracentrifugation sedimentation

Sedimentation velocity analysis was conducted at 20°C and 36,000 RPM using absorbance optics with a Beckman-Coulter XL-I analytical ultracentrifuge. Double sector cells equipped with quartz windows were used. The rotor was equilibrated under vacuum at 20 °C and after a period of ~1 hour at 20°C the rotor was accelerated to 36,000 RPM. Absorbance scans at 280 nm were acquired at 6 minute intervals for ~8 hours. Four pSTAT3 samples (2, 4, 8 and 16 ^M) were tested in AUC buffer (20 mM HEPES, 200 mM NaCl, 10 mM MgCl2, pH 7.0.). Experimental data were analyzed for protein purity, aggregates and oligomerization status by using c(s) distribution analysis as implemented in Sedfit v13.0b. The analysis showed that the purified pSTAT3 protein was a homogenous solution of pSTAT3 dimers (see FIG.2C).

Surface Plasmon Resonance (SPR) analysis

The in vitro binding affinity of the BBI-608 for the purified pSTAT3 was determined using Surface Plasmon Resonance (SPR).

The binding kinetics of the BBI-608 with pSTAT3 protein was measured using a Biacore T100. The purified pSTAT3 protein was immobilized on a CM5 chip by an amine coupling method. The CM5 sensor chip surface was treated by a 7 min activation using EDC/NHS, followed by a 7 min injection of pSTAT3 protein (100 μg/ml in 10 mM Sodium acetate pH 5.5). The surface was blocked by 1 M ethanolamine pH 8.5 for 7 min. 10 mM Glycine pH 2.7 was injected via 3-5 10 second pulses to wash off the non-covalently bound protein and to stabilize the baseline. The surface was again washed with PBS running buffer containing 5% DMSO, 3 mM EDTA, and 0.05% P20. A stock BBI-608 containing 100% DMSO was diluted with fresh 1X PBS to a working concentration of 50 μM. The BBI-608 was then injected over the reference and the protein flow cells. Each cycle consisted of association and dissociation of 120 seconds each and the flow rate was 60 μL/min. A solvent correction procedure was utilized to compensate for the use of DMSO in the experiment. The SPR analysis showed that the BBI-608 bound to purified pSTAT3 with a Kd of about 100 nM to 200 nM (FIG.2D). Inhibition of STAT3 DNA binding in vitro To investigate whether binding of BBI-608 to pSTAT3 inhibits pSTAT3's DNA binding activity, the purified pSTAT3 dimer was first incubated for 15 minutes with a radiolabeled probe comprising a STAT3 DNA binding site (see EXAMPLE I). The STAT3-DNA complex was then incubated with 1 μM of the BBI-608. The results shown in FIG.2E indicate that the BBI-608 decreased STAT3 DNA binding by 3-4 fold. As a comparison, a STAT3-SH2 domain binding phosphopeptide (P-pY-LKTK) that acts as a selective inhibitor of STAT3 dimerization also blocked pSTAT3-DNA binding completely at 2 mM. These data demonstrate that the BBI-608 acts directly on pSTAT3 to inhibit the binding of STAT3 to its cognate DNA binding site. EXAMPLE 3: Co-Crystallization of a Compound of the Disclosure with pSTAT3 bound to its cognate DNA binding site Co-Crystallization of pSTAT3/DNA/ Compound of the Disclosure The location and mode of binding of a Compound of the Disclosure, 2- acetylnaphtho [2,3-b] furan-4,9-dione (also referred to herein as BBI-608), was determined by X-ray crystallography. The purified pSTAT3 protein was concentrated by Amicon Ultra-15 centrifugal filter and co-crystallized with a DNA duplex comprising a STAT3 DNA binding site (Forward: 5’-AAGATTTACGGGAAATGC-3’ (SEQ ID No.: 6) and Reverse: 5’- TGCATTTCCCGTAAATCT-3’) (SEQ ID No.: 7) in SEC running buffer. The crystallization solution contained a final STAT3 protein concentration of about 5 mg/ml (i.e., a molar ratio of about 2:1 with respect to the concentration of the DNA duplex). The final concentration of the BBI-608 was 0.8 mM (i.e. a molar ratio of about a 10:1 with respect to the concentration of pSTAT3). pSTAT3 protein was pre-incubated with the BBI-608 for 2-3 hours before mixing with DNA duplex comprising the STAT3 DNA binding site. Crystals were then grown using the hanging drop method over a reservoir that contained 500 ^l of 0.4 M ammonium phosphate monobasic or 0.8 M sodium /potassium phosphate pH 5.0. pSTAT3/DNA/BBI-608 co-crystals were obtained using both conditions. The final cryoprotection solution contained 35% glycerol.

X-ray data collection and structure determination

Diffraction data were collected at the Cornell High Energy Synchrotron Source (CHESS) beamline A1 at 100 K with a CCD detector. Raw data were reduced and processed using HKL3000 at CHESS site. The complex structure was solved by molecular replacement using the program MOLREP, implemented in CCP4. The starting model was derived from published structure (Becker, Groner et al.1998) (PDB id 1BG1) with all solvent residue removed. A simple rigid-body refinement was sufficient to initiate refinement, with subsequent refinement and model building cycles performed using Refmac5 and Coot. The X-ray data (TABLE 2 A) and refinement statistics (TABLE 2B) are summarized below.

Figure imgf000051_0001

The overall architecture of the pSTAT3/BBI-608/DNA complex shown in FIG. 3A was similar to the published pSTAT3/DNA structure (see Ren et al., Biochemical and biophysical research communications 374, 1-5 (2008)). The dimeric pSTAT3 molecules, linked by the well-known reciprocal SH2-pY exchange, contact the DNA duplex by the previously recognized contact residues in the DNA binding domain which is constructed mainly of a series of beta strands. The linker domain, a region containing four separated long helices, was interposed between the DNA binding domain and the SH2 domain (FIG.3A). The BBI-608 was found within a pocket formed at the junction between the DNA binding domain and linker domain (FIG. 3A, inset, and FIG. 3B). This pocket was determined to be the hinge domain, since it acts as a pivot point between the upper domains (SH2 and linker) and lower domains of pSTAT3 (DNA binding and coil:coil (CC) domain). This hinge pocket was outlined by five residues from the DNA binding domain: a loop region from residue M331 to residue R335 plus P471, the second residue in the first helix of the DNA binding domain. M470 from the DNA binding domain was located at the base of the pocket and holds the BBI-608 upward in the overall STAT3 structure. The ceiling of the pocket was mainly composed of two residues from the last helix of the linker domain, D570 and K573. Residue T515 from the second helix of the linker domain was also involved in reordering the pocket surface, but it did not come in direct contact with BBI-608. The entrance of the hinge pocket was partially blocked by the DNA molecule. The only direct contact between the BBI-608 and the target DNA involved the nucleotide phosphate group on position +1 (G11) of the GAS consensus sequence (FIG. 3B). As shown in TABLE 3 below, multiple strong polar, hydrophobic, and hydrogen bond interactions are involved in the binding of BBI-608.

Figure imgf000053_0001

The planar shape of BBI-608’s naphthoquinone-furan moiety was well defined by the 2Fo-Fc electron density map (FIG. 3B). No density was observed for the acetyl group due to its flexibility. EXAMPLE 4: A Compound of the Disclosure inhibits STAT3 through an allosteric-like mechanism The conformational changes resulting from the binding of a Compound of the Disclosure to STAT3's hinge pocket were deduced by superimposing the structure of the pSTAT3-DNA complex bound by BBI-608 on the previously published structure of the drug free pSTAT3-DNA complex. Structural comparison of drug free pSTAT3-DNA and BBI-608 bound pSTAT3-DNA. The BBI-608 bound structure (dark gray) and drug-free structure (PDB: 1BG1, in light gray) and the monomeric core fragment STAT3 structure without DNA were superimposed by overlapping their coiled coil domain and DNA binding domain (see FIG.4). FIG.4A illustrates the direction of the domain shift induced upon drug binding (see arrows). FIG. 4B shows using a stick representation which residues within the hinge pocket interact with the BBI-608. FIG. 4C shows a top view of the overlapped structures that reveals the creation of a ~ 0.5Å gap between the SH2-SH2 dimer. FIG. 4D shows a 2Fo-Fc map of pY705’s phosphate group with residue R609 from another monomer (Gray mesh at 1.5σ). EXAMPLE 5: Determination of the stoichiometry and binding affinity of pSTAT3 to DNA in the presence and absence of a Compound of the Disclosure The affinity and stoichiometry of the binding of pSTAT3 to DNA in the presence and absence of a Compound of the Disclosure was determined using isothermal titration calorimetry (ITC). ITC experiments were conducted at 298 K on an ITC200 Microcalorimeter (Malvern). Purified pSTAT3 was extensively dialyzed against the ITC buffer (20 mM HEPES pH 7.0, 200 mM NaCl, 10 mM MgCl2, and 0.5 mM TCEP). The desired concentration of pSTAT3 was obtained by filtration through an Amicon Ultra-15 centrifugal filter. Lyophilized DNA oligos was directly dissolved in the ITC buffer and freshly annealed before the experiment. The DNA oligo used in the ITC assay was identical to that used in the crystallization assay. Pure DMSO or 10 mM BBI-608 diluted in 100% DMSO stock was added to the protein solutions. The final DNA and protein solutions contained 2% DMSO. The assay was set to titrate 200 µM double-strand DNA into 34 µM pSTAT3, with DMSO only or with 17 µM BBI-608. The binding affinity of pSTAT3 for DNA in the presence of DMSO was found to have a Kd of ~ 97 nM and the stoichiometry was 0.5, where one double stranded DNA molecule bound two pSTAT3 molecules (FIG. 5C). In the presence of the BBI- 608, the binding affinity of pSTAT3 to DNA had a Kd of ~ 20 μM, representing a 200- fold reduction. In the presence of BBI-608, the binding stoichiometry was 0.72, suggesting one double stranded DNA molecule bound 1.5 pSTAT3 molecules (FIG. 5D). Collectively, these data show quantitatively that BBI-608 reduced the affinity between pSTAT3 and DNA. EXAMPLE 6: DNA conformational changes induced by the Compound of the

Disclosure DNA conformational changes in the pSTAT3/DNA complex formation induced by a Compound of the Disclosure were detected using fluorescence polarization. Fluorescence labeled DNA oligos were purchased from IDT. The fluorescent label, Alexa Fluor 488 was attached only on the 5’ of the reverse 12-bp DNA strand (5’- ATTTCCCGTAAA-3’ (SEQ ID NO.: 4)). The forward 12-bp DNA strand was unlabeled (5’-TTTACGGGAAAT-3’ (SEQ ID NO.: 5)). The DNA oligos were dissolved in nuclease-free water and annealed before use. The fluorescence polarization assay buffer contains 17.6 mM HEPES, 176 mM NaCl, 8.8 mM MgCl2, 4.4 mM DTT, pH 7.0, 2% DMSO. A stock solution of 10 mM BBI-608 in 100% DMSO was used. All reactions and measurements were performed at room temperature. BBI-608 was first pre-incubated with pSTAT3, then double stranded DNA was added and incubated for 15 min, followed by the measurements taken by FilterMax F5 (Molecular Devices). The data fittings were performed using GraphPad Prism. The solution of preformed pSTAT3 and fluorophore-labeled DNA (fluo-DNA) complexes was titrated with the BBI-608. As the concentration of the BBI-608 increased, an increasing amount of free fluo-DNA was released from the pSTAT3/fluo- DNA complex, thus decreasing the total fluorescence polarization (FIG. 5E). Furthermore, upon titration by free fluo-DNA with pSTAT3, the total fluorescence polarization increased at a much lower rate when BBI-608 was present (FIG. 5F), demonstrating that pSTAT3/DNA complex formation was reduced in the presence of the BBI-608.

The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors at the time of filing to make and use the invention. The discussions as to what the evidence may suggest are intended solely to assist those skilled in the art to understand and thus make and use the invention in the best way known to the inventors at the time of filing. Nothing in this specification should be considered as limiting the scope of the present invention. All examples presented are representative and non-limiting. The above- described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

Claims

CLAIMS 1. A method for modulating STAT3 activity comprising binding one or more
amino acid residues in STAT3's linker domain.
2. The method of claim 1, wherein STAT3 is bound to a STAT3 DNA binding site.
3. The method of claim 1, wherein the binding to the linker domain comprises binding to at least one amino acid residue chosen from T515, D570, or K573 of SEQ ID NO: 1.
4. The method of claim 1, wherein the binding to the linker domain comprises binding to T515, D570, and K573 of SEQ ID NO: 1.
5. The method of any one of claims 1-4, comprising binding to one or more amino acid residues in a STAT3 DNA binding domain.
6. The method of claim 5, wherein the binding to the DNA binding domain
comprises binding to at least one amino acid residue chosen from M331, H332, P333, D334, R335, P471, or M470 of SEQ ID NO: 1.
7. The method of claim 5, wherein the binding to the DNA binding domain
comprises binding to M331, H332, P333, D334, R335, P471, and M470 of SEQ ID NO: 1.
8. The method of any one of claims 1-7, wherein the binding reduces STAT3
binding affinity for a STAT3 DNA binding site.
9. A method for modulating an activity of STAT3 comprising contacting one or more amino acid residues in STAT3's hinge pocket.
10. The method of claim 9, wherein STAT3 is bound to a STAT3 DNA binding site.
11. The method of claim 9 or claim 10, wherein the contact with the one or more amino acid residues in the hinge pocket comprises contacting one or more amino acid residues in STAT3's linker domain and DNA binding domain.
12. The method of claim 11, wherein the one or more amino acid residues in the DNA binding domain is chosen from M331, H332, P333, D334, R335, P471, or M470 of SEQ ID NO: 1.
13. The method of claim 11, wherein the one or more amino acid residues in the linker domain is chosen from T515, D570, or K573 of SEQ ID NO: 1.
14. The method of any one of claims 9-13, wherein the contact with the one or more amino acid residues in the hinge pocket induces a change in STAT3, said change comprising increasing the distance between the SH2 domains of the STAT3 and/or bending the minor groove of the STAT3 DNA binding site.
15. A method for modulating STAT3 activity in a cell comprising contacting one or more amino acid residues in STAT3's hinge pocket.
16. The method of claim 15, wherein the cell is a cancer stem cell.
17. A method for preventing or treating a disease or disorder associated with an aberrant STAT3 signaling pathway in a subject in need thereof comprising contacting one or more amino acid residues in STAT3's hinge pocket.
18. The method of claim 17, wherein the disease or disorder comprises a cancer, an autoimmune disease, an inflammatory disease, inflammatory bowel diseases, arthritis, autoimmune demyelination disorder, Alzheimer's disease, stroke, ischemia reperfusion injury, or multiple sclerosis.
19. The method of claim 18, wherein the cancer is lung cancer, colon cancer, rectal cancer, colorectal cancer, gastrointestinal cancer, esophageal cancer, small bowel cancer, gastroesophageal junction cancer, appendiceal cancer,
chondrosarcoma, adrenocorticoid cancer, laryngeal cancer, squamous cell carcinoma, angiosarcoma, leukemia, lymphoma, myeloma, brain cancer, ovarian cancer, melanoma, pancreatic cancer, gastric cancer, prostate cancer, breast cancer, liver cancer, renal cancer, myelodysplastic syndromes,
cholangiocarcinoma, endometrial cancer, neuroendocrine cancer, bladder cancer, mesothelioma, synovial sarcoma, thymic cancer, Desmoid tumor, or head and neck cancer.
20. The method of claim 18, wherein the cancer is a metastatic cancer, a cancer that is refractory to chemotherapy, a cancer that is refractory to radiotherapy and/or a cancer that has relapsed.
21. A method for modulating an activity of a non-druggable protein that has a linker domain, comprising binding one or more amino acid residues in the protein's linker domain.
22. The method of claim 21, wherein the non-druggable protein further comprises an effector domain and a binding domain.
23. The method of claim 22, wherein the binding domain comprises a nucleic acid binding domain.
24. The method of claim 23, wherein the binding to the linker domain reduces the non-druggable protein's affinity for its cognate nucleic acid binding site.
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