Claims:1. A pharmaceutically active compound comprises:

a plurality of small molecules as Wnt transcription inhibitors, wherein the plurality of small molecules target and inhibit key proteins of nuclear transcription complex to inactivate the Wnt signaling pathway in growth of a tumor, and

a macrocyclic/non-macrocyclic compound having tetrahydrofuran moiety sub-structure as a Wnt/b-catenin inhibitors to suppress the growth of the tumor,

wherein the macrocyclic compound involves in synthesis of tetrahydrofuran derivative through an iodocyclization reaction of resultant product from the reaction of R, R-tartaric acid, and a chiral starting material.

2. The compound of claim 1, further several isomeric lactones are synthesized from R, R-tartaric acid to produce tetrahydrofuran ring compound through iodocyclization reaction.

3. The compound of claim 1, wherein the macrocyclic/non-macrocyclic compound is a 14-membered ring furan- macrocyclic/non-macrocyclic compound with a single olefin geometry.

4. The compound of claim 1, wherein the tetrahydrofuran ring is selectively hydrolyzed and coupled with four amino acid derivatives to obtain four different macrocyclic precursors, numbered SM-108, SM-109, SM-110, and SM-111.

5. The compound of claim 4, wherein the five macrocyclic precursors are subjected to the ring-closing metathesis to produce four 14-membered ring macrocycle compounds numbered as SM-112, SM-113, SM-114, and SM-115. , Description:BACKGROUND OF THE INVENTION
A. Technical field
[0001] The present invention generally relates to the pharmaceutically active candidates against Wnt biocomplex, and more particularly, the identification of small molecules as Wnt/b-catenin pathway inhibitors. More specifically, the present invention relates to the identification of small molecules working at the Wnt Transcription Machinery, a method of synthesis of tetrahydrofuran-based macrocyclic and non-macrocyclic compounds, and the methods of preparation thereof.

B. Description of related art
[0002] The word Wnt is originated from a combination of “Winglass and Integrated”. The Wnt pathway is generally characterized as the canonical Wnt pathway and non-canonical Wnt/calcium pathway. The functioning of the Wnt pathway starts from the activation of cell surface receptors by the binding of a Wnt-protein ligand to a Frizzled family receptor. This activation then leads to passing on the signal inside the cell. It is well-known that the canonical Wnt pathway plays a central role in gene regulation. Typically, these processes are highly conserved, ranging from fruit flies to humans.

[0003] During the early days, the Wnt pathway is commonly known for its importance in carcinogenesis. Later, the Wnt pathway receives serious attention in embryonic development. In this process, the Wnt pathway seems to play a pivotal role in body axis, cell fate, cell proliferation as well as cell migration. All these factors are necessary for the accurate formation of important tissues including, bone, heart, and muscle. The role of the Wnt pathway in the embryonic development is discovered when genetic mutations in Wnt pathway proteins led to producing abnormal fruit fly embryos. The important role of this pathway is also discovered during tissue regeneration in adult bone marrow, skin, and intestine. Later, it is observed that the genes responsible for these abnormalities played a key role in breast cancer development in mice.

[0004] The Wnt/beta-catenin pathway is a family of proteins and involved in many vital cellular functions like stem cell regeneration and organogenesis. For example, Wnt activation is critical to the optimal maintenance of stem cells. In general, colorectal cancers show the evidence of Wnt signaling pathway activation and this is further associated with the loss of function of the tumor regulator APC. It is also known that the Wnt activation is commonly observed in breast, lung, and hematopoietic malignancies and it further contributes to tumor recurrence. The Wnt/beta-catenin pathway is also known to have the cross talks with Notch and Sonic Hedgehog pathways; this leads to serious implications for therapeutic intervention in cancers. One of the challenges dealing with the Wnt pathway is finding effective agents without damaging the system of normal somatic stem cell function in cellular repair and tissue homeostasis.

[0005] Currently, few existing patent references attempted to address the aforementioned problems are cited in the background as prior arts over the presently disclosed subject matter and are explained as follows.

[0006] A prior art US 10300109 B2 to Huw M. Nash, et al., entitled “Peptidomimetic macrocycles” disclose peptidomimetic macrocycles comprising a helix, such as an alpha helix, and methods of using such macrocycles for the treatment of disease such as cancer. In other aspects, the peptidomimetic macrocycle comprises an a,a-disubstituted amino acid, or may comprise a crosslinker linking the a-positions of at least two amino acids or at least one of said two amino acids may be an a,a-disubstituted amino acid. Further included is the targeting of components of the Wnt signaling pathway such as the Tcf4-/3-catenin complex.

[0007] Another prior art WO2011014973 to Daniel Obrecht, et al., entitled “Conformationally constrained, fully synthetic macrocyclic compounds” discloses a Conformationally restricted, spatially defined 12-30 membered macrocyclic ring systems of formulae Ia and Ib are constituted by three distinct molecular parts such as template A, conformation modulator B and bridge C. These macrocycles Ia and Ib are readily manufactured by parallel synthesis or combinatorial chemistry in solution or on a solid phase. They are designed to interact with a variety of specific biological target classes, examples being the agonistic or antagonistic activity on G-protein coupled receptors (GPCRs), ion channels, and signal transduction pathways. In particular, these macrocycles act as antagonists of the motilin receptor, the FP receptor and the purinergic receptors P2Y1, as modulators of the serotonin receptor of subtype 5-HT2B, as blockers of the voltage-gated potassium channel Kv1.3 and as inhibitors of the ß-catenin-dependent "canonical" Wnt pathway.

[0008] Though the above mentioned prior arts disclose a method of synthesis of pharmaceutically active 14 membered macrocyclic ring along with small molecules that act as a signal pathway inhibitor to suppress the growth of tumors, none of the prior art discloses the synthesis of the furan substituted 14 membered macrocyclic compounds. Therefore, there is a need for the synthesis of a new family of furan-macrocyclic compounds and their further utilization in the identification of novel small molecules functioning as the Wnt transcription inhibitors. Further, there is a need for an approach that is useful for interfering with tumor growth.

SUMMARY OF THE INVENTION
[0009] The present invention generally discloses the field of the synthesis of pharmaceutically relevant, small molecules as Wnt transcription inhibitors. Further, the present invention discloses the identification of small molecules working at the Wnt transcription machinery, a method of synthesis of tetrahydrofuran-based macrocyclic and non-macrocyclic compounds, and the methods of preparation thereof.

[0010] According to the present invention, the pharmaceutically active compound is developed to inhibit Wnt/beta (b)-catenin pathway, especially at the level of the nuclear complex between b-catenin and TCF (T-Cell Factor) or LEF (Lymphoid Enhancer Factor), and an approach which is useful to interfering with the tumor growth. In one embodiment, the pharmaceutically active compound comprises a plurality of small molecules and a macrocyclic/non-macrocyclic compound. In one embodiment, the small molecules act as canonical Wnt signaling pathway inhibitors. In one embodiment, the small molecules could inhibit the Wnt/beta (b)-catenin pathway to suppress the growth of the tumor.

[0011] In one embodiment, the small molecules target and inhibit the key proteins of nuclear transcription complex such as (b)-catenin, TCF (T-Cell Factor) or LEF (Lymphoid Enhancer Factor), and CBP (chronic bacterial prostatitis). The small molecules target and inhibit the key proteins of the nuclear transcription complex to inactivate the Wnt signaling pathway in the growth of tumors such as in breast cancer patients. In one embodiment, the Wnt/b-catenin signaling pathway is triggered by binding the Wnt ligand to the LRP-5/6 receptor (low-density lipoprotein receptor) and Frizzled receptors. In one embodiment, the generic structure of the furano-macrocycle is also shown as tetrahydrofuran moiety. With this goal, the invention has two objectives. In one embodiment, the first objective is to develop a practical and scalable synthesis of the scaffold. In one embodiment, the second objective is to obtain furan-macrocyclic compounds with the 14-membered ring upon the utilization of the tetrahydrofuran moiety.

[0012] In one embodiment, the macrocyclic/non-macrocyclic compound is synthesized with tetrahydrofuran moiety sub-structure as a Wnt/b-catenin inhibitors to suppress the growth of the tumor. In one embodiment, the macrocyclic compound involves in synthesis of tetrahydrofuran derivative through an iodocyclization reaction of resultant product from the reaction of R, R-tartaric acid, and a chiral starting material. In one embodiment, the synthesized macrocyclic/non-macrocyclic compound is a 14-membered ring furan- macrocyclic/non-macrocyclic compound with a single olefin geometry.

[0013] In one embodiment, several isomeric lactones are synthesized from R, R-tartaric acid to produce tetrahydrofuran ring compound through iodocyclization reaction. In one embodiment, wherein the tetrahydrofuran ring is selectively hydrolyzed and coupled with four amino acid derivatives to obtain four different macrocyclic precursors, numbered SM-108, SM-109, SM-110, and SM-111. In one embodiment, the five macrocyclic precursors are subjected to the ring-closing metathesis to produce four 14-membered ring macrocycle compounds numbered as SM-112, SM-113, SM-114, and SM-115.

[0014] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF DRAWINGS
[0015] The foregoing summary, as well as the following detailed description of the invention, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, exemplary constructions of the invention are shown in the drawings. However, the invention is not limited to the specific methods and structures disclosed herein. The description of a method step or a structure referenced by a numeral in a drawing is applicable to the description of that method step or structure shown by that same numeral in any subsequent drawing herein.

[0016] FIG. 1 shows a schematic diagram of the Wnt/beta (b)-catenin pathway in the absence and presence of the Wnt ligand in an embodiment of the present invention.

[0017] FIG. 2 shows four representative examples of bioactive natural products having one or more than one furan rings in the architectures in one embodiment of the present invention.

[0018] FIG. 3A shows the generation luciferase-based primary screening assay to identify Wnt-b-catenin inhibitors in one embodiment of the present invention.

[0019] FIG. 3B shows the luciferase-based screening data with SM-110, SM-111 (both precursor to macrocyclic compounds), and SM-114 and SM-115 in one embodiment of the present invention.

[0020] FIG. 3C shows the RT-PCR data related to genes, TCF, and LEF with small molecules SM-110 (open chain precursor) and SM-115 (macrocyclic compound) in one embodiment of the present invention.

[0021] FIG. 3Dshows the RT-PCR data with specific genes, SNAIL, CKS-2, and HEF-1, related to cell migration and cell invasion with small molecules SM-110 and SM-115 in one embodiment of the present invention.

[0022] FIG. 3E shows the RT-PCR data with a specific gene, VIMENTIN, related to cell metastasis and proliferation with small molecules SM-110 and SM-115 in one embodiment of the present invention.

[0023] FIG. 3F shows the RT-PCR data related to genes, Axin, b-catenin (also known as CTNBB-1), and APC with small molecules SM-110 (open chain precursor) and SM-115 (macrocyclic compound) in one embodiment of the present invention.

[0024] FIG. 4A shows the western blots related to phospho-b-catenin, b-catenin, TCF-1/TCF-7, after treatment with IWR-1, SM-110, and SM-115 in one embodiment of the present invention.

[0025] FIG. 4B shows the western blots related to AXIN-2, LEF, CBP, TCF-1, and cyclin D1 after treatment with IWR-1, SM-110, and SM-115 in one embodiment of the present invention.

[0026] FIG. 5 shows the RNA sequencing data with cell migration gene set upon treatment with small molecules, IWRI (positive control from literature), SM-61 (open structure, precursor to macrocycle), and SM-66 (macrocyclic compounds obtained from SM-61) in different embodiments of the present invention.

[0027] FIG. 6A shows the effect of small molecules, IWR-1, SM-110, and SM-115 on the formation of tumorspheres (G2; 2nd generation) from an established cancer cell line, HCT116 in one embodiment of the present invention.

[0028] FIG. 6B shows the RT-PCR for TCF4 and LEF gene on the formation of tumorspheres (G2; 2nd generation) from the established cancer cell line, HCT116 in the presence of, IWR-1, SM-110 and SM-115 in one embodiment of the present invention.

[0029] FIG. 6C shows the RT-PCR for cell migration and invasion genes, SNAIL and HEF1 on the formation of tumorspheres (G2; 2nd generation) from the established cancer cell line, HCT116 in the presence of, IWR-1, SM-110, and SM-115 in one embodiment of the present invention.

[0030] FIG. 6D shows the RT-PCR for the metastasis gene, VEMENTIN on the formation of tumorspheres (G2; 2nd generation) from the established cancer cell line, HCT116 in the presence of, IWR-1, SM-110, and SM-115 in one embodiment of the present invention.

[0031] FIG. 6E shows the RT-PCR for the stemness genes, CD44, and CD133 on the formation of tumorspheres (G2; 2nd generation) from the established cancer cell line, HCT116 in the presence of, IWR-1, SM-110, and SM-115 in one embodiment of the present invention.

[0032] FIG. 7A shows the effect of small molecules, IWR-1, SM-110, and SM-115 on the formation of tumorspheres (G2; 2nd generation / patient 1) from Indian patient 1 buccal mucosa in one embodiment of the present invention.

[0033] FIG. 7B shows the effect of small molecules, IWR-1, SM-110, and SM-115 on the formation of tumorspheres (G2; 2nd generation/patient 2) from Indian patient 2 buccal mucosa in one embodiment of the present invention.

[0034] FIG. 7C shows the RT-PCR for TCF4 and LEF gene on the formation of tumorspheres (G2; 2nd generation / patient 2) in the presence of, IWR-1, SM-110, and SM-115 in one embodiment of the present invention.

[0035] FIG. 7D shows the RT-PCR for the metastasis gene, VEMENTIN on the formation of tumorspheres (G2; 2nd generation / patient 2) in the presence of IWR-1, SM-110, and SM-115 in one embodiment of the present invention.

[0036] FIG. 7E shows the RT-PCR for cell migration and invasion genes, SNAIL and HEF1 on the formation of tumorspheres (G2; 2nd generation / patient 2) in the presence of IWR-1, SM-110, and SM-115 in one embodiment of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS
[0037] A description of embodiments of the present invention will now be given with reference to the Figures. It is expected that the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive.

[0038] According to the present invention, an improved and effective pharmaceutically active small molecules as Wnt/b-catenin pathway inhibitors and a method of synthesis of furan-based, novel macrocyclic compounds thereof, are disclosed. The small molecules work at the Wnt transcription machinery as the inhibitors of cancer cell migration and metastasis. Referring to FIG. 1, a schematic diagram of the Wnt/b-catenin pathway in the absence and presence of the Wnt ligand, according to one embodiment of the present invention. In one embodiment, the pathway is activated by the binding of a Wnt-protein ligand to a Frizzled family receptor, which then transmits the biological signal inside the cell. The Wnt/b-catenin signaling pathway is first identified for its role in carcinogenesis, then for its function in embryonic development, including body axis, cell fate specification, cell proliferation, and cell migration.

[0039] In one embodiment, the Wnt/b-catenin signaling pathway is triggered by binding the Wnt ligand to the LRP-5/6 receptor (low-density lipoprotein receptor) and Frizzled receptors. Following this activation, it leads to the recruitment of the complex, comprising many proteins, such as Axin, GSK-3 beta, CK1, APC as shown in FIG. 1. This cytosolic protein complex plays a central role in maintaining the phosphorylation of beta-catenin; which is then subjected to protein degradation machinery through ubiquitination. An imbalance further leads to the accumulation of un-phosphorylated beta-catenin in the cytosolic region which then migrates to the nucleus. At this stage, beta-catenin plays a pivotal role through participating in interactions with T-cell specific factor (TCF) / lymphoid enhancer-binding factor (LEF) and the other co-activators, for example, CBP (i.e. CREB-binding protein, also known as CREBBP or CBP). This whole complex machinery leads to activating Wnt target genes, such as c-Myc, cyclin D1, and Cdkn1a.

[0040] Referring to FIG. 2, four representative examples of bioactive natural products having one or more than one furan rings in the architectures, according to one embodiment of the present invention. In one embodiment, the present invention discloses the synthesis of a new family of furan-macrocyclic compounds and their further utilization in the identification of novel small molecules functioning as the Wnt transcription inhibitors. The synthesis methodology utilizes several examples of bioactive natural products as well as drugs that are known to have the tetrahydrofuran ring moiety in their complex architectures. Due to their nature, these sub-structures are classified as the “privileged” moiety. In one embodiment, the bioactive natural products include eribulin or compound 1, amphidinolide E or compound 2, amphidinolide F or compound 3, and haterumalides or compound 4. In one embodiment, eribulin is a well-known anti-cancer drug derived from the truncated structure of a marine natural product contains several tetrahydrofuran rings in its complex macrocyclic architecture.

[0041] In one embodiment, the synthesis of furano-macrocyclic compounds 6 having the tetrahydrofuran moiety 5 as the privileged sub-structure is disclosed in Scheme 1. The practicality and ease of the synthesis of these relatively complex furan-based macrocyclic compounds are one of the unique features of the present invention. In particular, screening of these macrocyclic as well as their precursors, search for Wnt/b-catenin inhibitors with special emphasis on the modulation of nuclear complex deal with multiple nuclear protein-protein interactions/DNA-protein interactions. Due to the ease of their synthesis, these compounds could also be subjected to extensive medicinal chemistry studies for further enhancing their biological potential. The possibility of developing a large scale, in a time-efficient manner, is also an attractive feature of these types of small molecules.

A. Synthesis Planning of Furan-Macrocyclic Compounds and Execution:

[0042] The fascinating architectures of many bioactive natural products can serve as an excellent starting point to building a tetrahydrofuran ring having a 14-membered macrocyclic ring-derived chemical toolbox, hunt for small molecule modulators of complex protein-protein interactions, and in other, selected signaling pathways. With our continuous interest in building a toolbox having compounds with different types of natural product-inspired macrocyclic rings, the present invention is started with the “stereo-defined tetrahydrofuran moiety” 6 as highlighted in Scheme 1. In one embodiment, the generic structure of the furano-macrocycle is also shown as tetrahydrofuran moiety 5.

[0043] With the goal of obtaining the stereo-defined tetrahydrofuran moiety 5, the present invention has two objectives. In one embodiment, the first objective is to develop a practical and scalable synthesis of the scaffold 13. In one embodiment, the second objective is to obtain furan-macrocyclic compounds with 14-membered ring 15 upon the utilization of the tetrahydrofuran moiety 5. In one embodiment, the presence of an amino acid moiety embedded in the macrocyclic ring could easily explore the chiral side chain of the amino acid moiety, for example, 15a, 15b, 15c, and 15d numbered as SM-112, SM-113, SM-114, and SM-115 respectively. In an exemplary embodiment, the key objective of the present invention is to further explore the value of these compounds in finding the inhibitors of the Wnt/b-catenin pathway. Upon the identification of functional chemical probes, the value of these hits are explored for functioning as the inhibitors of tumor cell migration/metastasis and cell invasion.

[0044] Referring to Schemes 2-4, the present invention utilizes S,S-tartaric acid/R,R-tartaric acid for esterification, reduction followed by monobenzylation, and oxidation. In one embodiment, the oxidation produces compounds 7 as shown in Scheme 2 in simple transformations (known in the literature). In a number of a few steps going through the key intermediate compounds 8 and 9, two corresponding lactones, 10a (major) and 10b (minor) are produced. As shown in Scheme 3, the major isomer of lactone 10a is then converted to compound 11. The compound 11 is then subjected to key iodocyclization, giving the crucial tetrahydrofuran derivative compound 12. At this stage, the product is thoroughly characterized by 1H-NMR, 2D-NMR, 13-NMR, and MS analysis; in particular, the stereochemistry of all the four chiral functional groups. This compound is led to obtain the desired tetrahydrofuran ring compound 13 as shown in Scheme 3. Scheme 4 shows the next step that involves the selective deprotection of the primary –OH group. In one embodiment, the amino acid is commercially available inexpensive enantioenriched building blocks. The amino acid/derivatives are widely present in several macrocyclic natural products as well as bioactive synthetic macro cycles. Hence, the aminoacid derived building blocks are incorporated into 14-membered macorcyclic skeleton compound 14 as a key diversity site. In one embodiment, the compounds 14 are numbered as SM-108 (L-Valin), SM-109 (L-isoleucine), SM-110 (L-Leucine), and SM-111 (L-Phenyl alanine). Finally, upon subjection to the ring-closing metathesis as the key reaction, the desired furano-macrocyclic compounds 15 are obtained. In one embodiment, the compounds 15 are numbered as SM-112 (L-Valin), SM-113 (L-isoLeucine), SM-114 (L-Leucine), and SM-115 (L-Phenyl alanine) as shown in Scheme 4 and 5. In all cases, the trans olefin geometry is obtained in the ring-closing metathesis reaction. All the products are thoroughly purified and well-characterized by MS and 1D- and 2D-NMR studies.

B. Biological Applications Using Substituted Furan-based Macrocyclic Compounds and their Acyclic Precursors:

1. Luciferase Assay (FIGs. 3A-3B):

Wnt Reporter Activity Assay:

[0045] Wnt Reporter Activity Assay is used in a 96 well format to identify active small molecules that have the potential in modulating modulate the Wnt signaling pathway. The LEADING LIGHT Wnt Reporter Assay Starter Kit is obtained from Enzo Life Sciences (cat. no. ENZ-61001-0001). The system contains an engineered 3T3 mouse fibroblast cell line, which expresses the firefly luciferase reporter gene under the control of Wnt-responsive promoters (TCF/LEF). Wnt target genes that contain consensus TCF/LEF binding element, which could be transcriptionally induced by the Canonical Wnt signaling pathway.

Protocol:

[0046] In one embodiment, the engineered 3T3 mouse fibroblast cells are seeded into 96 well plates or 3T3 at a density of 10,000 cells/well. After 24 hours, cells are treated with the small molecules at a final concentration of 10µM re-suspended in the assay medium. After the 24 hours post-treatment, the firefly luciferase reagent is added to the cells and it is then subjected to immediately for reading the luminescence using a plate reader (Promega E1910). The top and bottom reads of the plate are taken and analyzed as the ratio of firefly luciferase and renilla luciferase. The Wnt reporter activity is plotted as a sample versus luciferase units that also include positive and negative controls.

2. RT-PCR Studies (FIGs. 3C-3F):

[0047] The quantitative Reverse transcription-polymerase Chain Reaction approach is used to understand the role of active small molecules on the Wnt/b-catenin pathway genes. In particular, the present invention focuses on the study of genes that are central to nuclear multi-protein transcription complex (i.e. b-catenin, LEF, TCF, and CBP) and control the b-catenin-mediated transcription machinery. The RT-PCR analysis shows a significant down-regulation of TCF4 and LEF genes as shown in FIG. 3C.

[0048] Typically, the Wnt/b-catenin signaling pathway is actively involved in various biological processes including cell proliferation, metastases, migration, and invasion. In one embodiment, the present invention further confirmed the effect of small molecules (SM-110 and SM-115) on cell proliferation, metastases, migration and invasion. Several candidate genes related to these pathways are examined and the expression of genes upon exposure is measured. The analysis of RT-PCR data clearly shows that SM-110 and SM-115 significantly reduce the expression of CCND1, Vimentin and HEF1, CKS2, and SNAIL genes 24h post-treatment as shown in FIG. 3D. The results suggest that small molecule SM-110 and SM-115 inhibits the Wnt/b-catenin signaling and its downstream effects in HCT116 cells. In addition, the data with the Vimentin gene is also shown in FIG. 3E.

[0049] In particular, the present invention focuses on the genes, such as, AXIN [36] and APC [37] that are involved in the protein destruction complex. Further, as shown in FIG. 3F, the RT-PCR study indicates that AXIN and APC genes are not affected by small molecules SM-110, SM-115, and the positive control, IWR-1 as shown in FIG. 3F. It appears that as with the positive control IWR-1, the small molecules, SM-110, and SM-115 stabilize the b-catenin protein destruction complex thereby inhibiting the b-catenin translocation to the nucleus.

[0050] The quantitative PCR (QuantStudio 5 Real-Time PCR System, USA) is performed using the STB GreenTM Premix Ex Taq TM II according to the manufacturer’s instruction (One-Step TB Green™ PrimeScript™ RT-PCR Kit II, USA). The canonical Wnt signaling pathway-related gene specific primers are shown below:

CCND1:
5'-ATGCCAACCTCCTCAACGAC-3'
Reverse primer? Let me get it from Anusha

VIMENTIN:
5'-TCTACGAGGAGGAGATGCGG-3'
5'-GGTCAAGACGTGCCAGAGAC-3

SNAIL2:
5'-ACCACTATGCCGCGCTCTT-3'
Reverse primer? Let me get it from Anusha

CKS2:
5’-CGCTCTCGTTTCATTTTCTGC-3’,
5’-TGGAAAGTTCTCTGGGTAACATAACA-3’

HEF1:
5'-CATAACCCGCCAGATGCTAAA-3'
5'-CCGGGTGCTGCCTGTACT-3'

CTNNB1:
5'-TCTGAGGACAAGCCACAAGATTACA-3'
5'-TGGGCACCAATATCAAGTCCAA-3'

APC:
5'-CATGATGCTGAGCGGCAGA-3'
5'-GCTGTTTCATGGTCCATTCGTG-3'

AXIN2:
5'-GAGTGGACTTGTGCCGACTTCA-3'
5'-GGTGGCTGGTGCAAAGACATAG-3'

TCF4:
5'-CTGCCTTAGGGACGGACAAAG-3'
5'-TGCCAAAGAAGTTGGTCCATTTT-3'

LEF1:
5'-AATGAGAGCGAATGTCGTTGC-3'
5'-GCTGTCTTTCTTTCCGTGCTA-3'

RNA isolation:

[0051] 0.5x106 HCT116 cells were cultured in a 6 well plate and cells are exposed to DMSO, IWR1, and small molecules (SM-61 and SM-66) for 24 h. RNA extraction is performed using Nucleo spin RNA isolation kit according to manufacturer protocol (MACHEREY-NAGEL GmbH & Co. KG). The quality and concentration of RNA are measured by nanodrop.

cDNA synthesis:

[0052] Total RNA is converted to cDNA using the PrimeScript 1st strand cDNA Synthesis kit (Takara, USA). The reaction composition includes 3 µg of total RNA, 1 µL of Oligo dT Primers, 1 µL dNTPs and 7 µL of RNAase freewater mixed in a polymerase chain reaction (PCR) tube. The Reverse transcriptase enzyme is added at the end and incubated at 65°C for 10 min, followed by cooling at 4 °C.

3. Western Blots (FIGs. 4A-4B):

Cell seeding and compound treatments:

[0053] HCT116 cells (ATCC Cat. No. CCL-247) representative of colorectal carcinoma are seeded into 6 well plates (cell seeding density: 0.3 million cells/well). After 24 hrs, the cells are treated with the compounds to achieve a final concentration of 10.0 µM along with DMSO and the positive control. After the 24 hrs post-treatment, the cells are harvested for protein expression analysis by western blotting.

Whole cell lysate preparation:

[0054] The treated cells are lifted and washed with PBS buffer. Then the cells are lysed used RIPA buffer (Sigma, Cat. No. R0278) supplemented with 1X protease inhibitor cocktail (Sigma, cat. no. S8820), 100µM Sodium ortho-vanadate (Sigma, cat. no. S6508), 50mM ß-Glycerophosphate (Sigma, Cat. No. G9422), 1mM 1,4-Dithiothreitol (Sigma, Cat. No. 10197777001). The lysis step is performed on ice for 30min with a gentle vortex at regular intervals. The lysed cell suspension is centrifuged at 14000 rpm for 10 min and supernatant is collected and quantified by bicinchoninic acid method (Pierce BCA Assay Kit, Thermo scientific, Cat. No. 23227).

Western blotting:

[0055] 50µg of total protein is used for separation on 10% SDS-PAGE; transferred to PVDF membrane at 100V, 2hrs. The blot is blocked with a 7% non-fat milk solution for 1hour. The primary antibody is added to the blots and incubated overnight at 4 0C under the rocking condition. The horseradish peroxidase (HRP) tagged secondary antibody is added and incubated at room temperature under rocking conditions. All the antibodies are procured from Cell Signalling Technologies. HRP-tagged GAPDH antibody (Santacruz, cat. no. sc-47724) is used as the loading control probing agent. The blots are developed with Femto LUCENT PLUS-HRP (G-Biosciences, cat. no. 786003). The results are captured using iBright Western blot imaging system (Thermoscientific, iBright CL1000 Imaging system). The imaging details are shown in FIGs. 4A and 4B.

4. RNA Sequencing Studies (FIG. 5):

Methods:

RNA Sequencing:

[0056] The total RNA (300 ng) is utilized for preparing RNA libraries using NEB ultra RNA library kit (NEB #E7770S/#E7775S). The mRNA is fragmented by the enzyme and then converted to cDNA according to the literature protocol. The cDNA is end-repaired and further purified using Ampure XP beads. The cleaned DNA is adapter-ligated and also purified using Ampure XP beads. These adapter-ligated fragments are then subjected to 12 cycles of PCR using primers provided in the kit. The PCR products are purified using Ampure XP beads. The quantification and size distribution of the prepared library is determined using Qubit flourometer and Agilent Tape station D1000 Kit (Agilent Technologies) according to the manufacturer’s instructions.

Data Analysis:

[0057] The transcriptome libraries (mRNA) are constructed using the NEB adapters and are sequenced on Illumina HiSeq at 150 nt read length using the paired-end chemistry. The raw data obtained is then processed for the low quality bases and adapters contamination. The raw reads subject to contamination (structural RNA/low complexity sequences) removal by mapping with bowtie 2-2.2.1. The decontaminated data set is mapped to the human genome [hg19] using hisat 2-2.1.0. Reads mapping to genes [hg19 gene list GTF] are counted using the feature count module of sub-reads package. The read counts are normalized in DESeq2-3.5, and subject to differential expression analysis.

Differential Gene Expression Analysis:

[0058] Significantly affected differentially expressed genes are selected based on 1.5x fold change, and then adjusted P-value of 0.05. Higherchial clustering heatmaps are created for the differentially expressed genes, using heatmap.2 function in R. Euclidian distance is used to calculate the distance between genes (rows) and samples (columns). As shown in Figure, different gene clusters could be distinguished for SM-66, SM-61, and IWR-1 based on their gene expression pattern in response to small molecules.

Results:

Differential Gene Expression:

[0059] HCT116 cells are exposed to 10 µM SM-61, SM-66, and IWR-1 for 24 h and then subjected to RNA sequencing analysis. To obtain an impression of the number of genes affected per treatment, the genes are selected using the criteria =2-fold up- or downregulation with adjusted p-value <0.05.

Pathway Analysis:

[0060] In order to seek more insight into the mechanism of action of SM61, SM66, and IWR1, the differentially expressed genes are analyzed for over-representation of the genes representative of WNT dependent biological processes. In one embodiment, the gene sets are selected from Molecular signature databases. As shown in FIGs. 6A-6F, most of the genes down-regulated by SM-66 and SM-61 are involved in WNT dependent biological processes including EMT, stemness, metastasis, cell migration, and invasion. The effect is more pronounced with SM-66 than SM-61 and it is not observed with IWR-1.

5. Effect on Tumorspheres Obtained from HCT 116 (FIGs. 6A-6E) and Indian Patient Buccal Mucosa (FIGs. 7A-7E):

Cell culture and tumorsphere formation:

[0061] The colorectal HCT116 cell lines are cultured in DMEM medium with 10% fetal bovine serum (FBS) and 5% antibiotics. For the tumorsphere formation, 1300 single cells per well are cultured in ultra-low attached 24-well plates with tumorsphere medium (Promo cell USA) with 2% penicillin–streptomycin. Cells are incubated for 7-10 days at 37 °C and 5% of CO2. The spheres are observed using an inverted microscope and Floid imaging system (Life sciences technologies, USA).

Patient derived tumorspheres:

[0062] Tissue biopsy samples are cleaned with PBS buffer, treated with antibiotic solution and necrotic or dead tissue is removed. Then, clean tissue samples are subjected to enzymatic digestion using collagenase and trypsin. After digestion, cells are transferred in DMEM medium with F12 and B27 and cultured for primary cancer cells (PCCs). After characterization, 1300 PCCs per well are seeded in 24 well ultra-low attachment plates with tumorsphere medium and incubated for 7-10 days at 37 °C and 5% of CO2 for deriving patient-derived tumorspheres. The spheres are captured using an inverted microscope and a Floid imaging system (Life sciences technologies).

Tumorsphere formation (from HCT116) in the presence of small molecules (FIG. 6A):

[0063] To check the effect small molecules on tumorspheres formation, the first and second generation HCT116 tumorsphere are treated with the positive control IWR1 (and in some cases, XAV 939) and small molecules SM-61 and SM-66 for 10 days. For primary generation spheres, the HCT116 cells (1300 cells/well) are seeded in 24 well ultra-low attachment plates with tumorsphere medium and then exposed to small molecules on the same day of initiation. After 7 days, the first generation (G1) spheres are collected and further re-seeded 1300 primary spheres for second-generation (G2) sphere formation. The effect of small molecules on second-generation spheres is measured by an inverted microscope and Floid image system at different time intervals and the images were recorded as appropriate. The RT-PCR data with genes, LEF and TCF4 are shown in FIG. 6B (down regulation of these genes) and FIG. 6C shows the RT-PCR analysis with two genes related to cell migration and invasion – SNAIL and HEF1 (note – significant down-regulation of HEF1 is observed); FIGs. 6D and 6E show the RT-PCR data with the genes, VIMNTIN (related to metastasis), CD44, and Cd133 (related to stemness). Significant downregulation of genes, VIMENTIN, and CD44 is noticed from the RT-PCR studies.

Patient-derived Tumorsphere formation in the presence of small molecules (FIG. 7A-7B):

[0064] To check the effect of small molecules on patient-derived tumorspheres formation, the first and second generation patient-derived tumorspheres of two Indian patients Buccal Mucosa are formed. The patient-derived tumorspheres are then exposed to positive control IWR-1 and SM-66 for 10 days. For the first-generation spheres, the PCCs are seeded in 24 ultra-low attachment plates with the tumorsphere medium. After 7 days, the first-generation spheres (G1) are collected and further re-seeded to single spheres for the second-generation (G2) sphere formation and then exposed to small molecules. The effect of small molecules on second-generation spheres is measured by an inverted microscope and a Floid image system record images at different time intervals.

[0065] As performed with HCT cell lines, several genes are analyzed on the formation of 2nd generation tumorsphere in the presence of small molecules SM-110, SM-115 and IWR-1. The genes selected for this study are related to Transcription machinery (TCF4 and LEF), metastasis (Vimentin gene), migration and cell invasion (SNAIL, HEF1, CKS2 gene) and stemness (CD44 and CD133). These results are shown in FIGs. 7C-7E.

[0066] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. It should be understood that the illustrated embodiments are exemplary only and should not be taken as limiting the scope of the invention.

[0067] The foregoing description comprise illustrative embodiments of the present invention. Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only, and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Merely listing or numbering the steps of a method in a certain order does not constitute any limitation on the order of the steps of that method. Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings in the foregoing descriptions. Although specific terms may be employed herein, they are used only in generic and descriptive sense and not for purposes of limitation. Accordingly, the present invention is not limited to the specific embodiments illustrated herein.