Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/ß-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/ß-catenin signaling within CSCs. Disruption of CBP-ß-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.

ICG-001, a novel small molecule regulator of TCF/beta-catenin transcription.

Inherited and somatic mutations in the APC gene, a human tumor-suppressor, occur in a large percentage of colon cancers, leading to elevated levels of nuclear beta-Catenin, and to activation of TCF/beta-Catenin-responsive genes including cyclin D1 and c-myc. To identify small molecule antagonists of this pathway, we screened transformed colorectal cells with a secondary structure-templated chemical library, in search of compounds that attenuated a TCF/beta-Catenin-responsive reporter gene. From this library we selected ICG-001 (IC50=3 microM) as a lead compound. Design and synthesis of the chemical library and some preliminary biological evaluation is described.

A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected].

Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of beta-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a beta-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates beta-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer.

Chemogenomics with peptide secondary structure mimetics.

There is increasing evidence that redox regulation of transcription, particularly activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB), is important in inflammatory diseases. Human thioredoxin (TRX), a member of the oxidoreductase superfamily, was initially identified, as a factor which augments the production of interleukin-2 receptor alpha (IL-2R alpha) in human T-cell lymphotropic virus type 1 (HTLV-1) infected patient T-cells. Substrates for the redox activity of TRX bind the active site cleft in extended strand structure. The rapid generation of high numbers of peptide secondary structure mimetics through solid-phase synthesis is a key technology for the identification of pharmaceutical leads based on such protein-peptide interactions. In this manuscript, we describe a chemogenomic approach utilizing an extended strand templated library to develop small molecule inhibitors to validate oxidoreductase molecular targets in a murine asthma model.

Chemogenomic identification of Ref-1/AP-1 as a therapeutic target for asthma.

Asthma is characterized by an oxidantantioxidant imbalance in the lungs leading to activation of redox-sensitive transcription factors, nuclear factor kappaB (NF-kappaB), and activator protein-1 (AP-1). To develop therapeutic strategies for asthma, we used a chemogenomics approach to screen for small molecule inhibitor(s) of AP-1 transcription. We developed a beta-strand mimetic template that acts as a reversible inhibitor (pseudosubstrate) of redox proteins. This template incorporates an enedione moiety to trap reactive cysteine nucleophiles in the active sites of redox proteins. Specificity for individual redox factors was achieved through variations in X and Y functionality by using a combinatorial library approach. A limited array (2 x 6) was constructed where X was either NHCH(3) or NHCH(2) Ph and Y was methyl, phenyl, m-cyanophenyl, m-nitrophenyl, m-acetylaniline, or m-methylbenzoate. These analogs were evaluated for their ability to inhibit transcription in transiently transfected human lung epithelial A549 cells from either an AP-1 or NF-kappaB reporter. A small-molecule inhibitor, PNRI-299, was identified that selectively inhibited AP-1 transcription (IC(50) of 20 microM) without affecting NF-kappaB transcription (up to 200 microM) or thioredoxin (up to 200 microM). The molecular target of PNRI-299 was determined to be the oxidoreductase, redox effector factor-1 by an affinity chromatography approach. The selective redox effector factor-1 inhibitor, PNRI-299, significantly reduced airway eosinophil infiltration, mucus hypersecretion, edema, and IL-4 levels in a mouse asthma model. These data validate AP-1 as an important therapeutic target in allergic airway inflammation.

Design, synthesis, and application of peptide secondary structure mimetics.

The secondary structure peptidomimetic approach is a rational way to develop novel nonpeptide pharmaceutical agents based upon biologically significant proteinaceous leads. A part of this approach elaborated in this laboratory over the past ten years is reviewed along with the recent developments in this field.

Design, synthesis, and evaluation of opioid analogues with non-peptidic beta-turn scaffold: enkephalin and endomorphin mimetics.

We have identified a mu-selective opioid receptor agonist without a cationic amino group in the molecule from libraries of bicyclic beta-turn peptidomimetics. The biologically active conformation of the lead is proposed to mimic an endomorphin type III 4 --> 1 beta-turn conformation.