Targets and Methods for Developing Therapeutics
Small Molecule Inhibitors of MicroRNA miR21
Micro RNAs are non-coding RNAs that appear to regulate gene expression. Numerous microRNAs are implicated in various diseases, including cancer, and provide new targets for therapeutic intervention.
Researchers at The Wistar Institute have developed a high-throughput screening (HTS) assay to identify inhibitors of oncogenic microRNAs. Using chemical libraries and the HTS assay, Wistar scientists (in collaboration with Dr. Alex Dieters at North Carolina State University) were able to identify several compounds capable of reducing the expression of the microRNA miR-21. This assay system may be used to find activators or inhibitors of other microRNAs.
Using the results of the HTS assay, the researchers designed and synthesized group of small molecules that selectively inhibit the microRNA miR21. MiR-21 functions as an anti-apoptopic agent in cancer cells and elevated levels of miR-21 has been associated with breast, ovarian, lung, and brain cancers and with heart failure. These compounds may be particularly useful in combination with low doses of standard chemotherapeutic drugs. Additional studies to demonstrate the in vivo efficacy of these compounds are planned.
Cancer Therapeutics Based on Novel Cyclopamine Analogs
Researchers at The Wistar Institute and The University of Pennsylvania have developed novel cyclopamine analogs with high potential as anti-cancer agents. Cyclopamine is a plant-derived alkaloid that is an effective inhibitor of the sonic hedgehog-GLI (SHH) signaling pathway which has been implicated in the development of a variety of cancers, including lung, prostate, and breast cancer. Cyclopamine has been demonstrated to reduce cancer cell proliferation, and to promote apoptosis in vitro and in vivo. However, cyclopamine is difficult and expensive to synthesize, limiting its usefulness as a therapeutic agent. Wistar and Penn researchers have developed a simple method for synthesizing cyclopamine analogs. Several of these analogs have been demonstrated to be effective inhibitors of sonic hedgehog signaling in vitro.
Design of Novel Molecules That Regulate Telomerase
We used the 3-dimensional structure of the catalytic subunit of telomerase to identify novel target binding sites essential for telomerase ribonucleoprotein assembly and activity. Telomerase adds multiple identical repeats of DNA (telomeres) to the 3’-end of eukaryotic chromosomes thus providing the genomic stability required for cell survival. There is now clear evidence that links telomerase to both cancer and aging. For example 90% of human cancers show high levels of activity of this enzyme when such activity is absent in most healthy tissues. The absence of telomerase activity (in adults) in healthy tissues leads to loss of ~50-100 bases of telomeric DNA with every cell division. When telomeres reach a critically short length, cells enter a permanent state of dormancy to prevent genomic instability, a process known as senescence, the hallmark of aging. Our goal is to identify compounds that regulate telomerase function that can be used to combat cancer and age related diseases.
Design of Novel Molecules that Regulate Sirtuins
Researchers at The Wistar Institute have identified and characterized a novel target binding site involved in regulating the activity of Sir2 proteins (or Sirtuins, the class III family of histone deacetylases, silent information regulator 2). Sirtuins deacetylate numerous cell proteins, thereby stimulating diverse biological functions related to metabolism and cell growth. Their activity has been linked to increased lifespan and calorie-associated effects on longevity. Compounds that target Sirtuins show promise in the treatment of diabetes, and diseases of degeneration and aging. Wistar scientists have identified and characterized a new molecular binding target, the nicotinamide binding site, which is expected to enable the development of new classes of small molecule Sirtuin modifiers. Experimental studies have validated the role of this binding site in the regulation of Sirtuin activity. Small-molecule compounds that target this site can act either as inhibitors of Sirtuins, or as effective Sirtuin activators, influencing a range of biological functions including DNA regulation, programmed cell death, neuronal protection, insulin signaling, and fat mobilization.
Suppression of Cell-Mediated Immunity by Down-regulation of Interleukin-12
The cytokine interleukin-12 (IL-12), which is produced during the immune response to a variety of stimuli, is critical for the development of cell-mediated immunity. Among its various functions in vivo, IL-12 is a potent inducer of interferon-gamma production by T lymphocytes and natural killer (NK) cells and is a co-factor in the mitogenic stimulation of these cell types. However, inappropriate cell-mediated immune responses, often accompanied by over-expression of IL-12, have been identified as having a key role in the development of a variety of autoimmune disorders, such as rheumatoid arthritis, multiple sclerosis, graft-versus-host disease, diabetes mellitus, systemic lupus erythematosus (lupus), and Crohn's disease. Reduction of IL-12 levels, for example by administration of antibodies to IL-12, has been effective in controlling autoimmune diseases in animal models.
Researchers at the Wistar Institute have determined that blockage of some complement receptors, including CD46 (the cellular receptor for measles virus) and CR3, can down-regulate production of IL-12 by monocytes. These findings support the well-known suppression of cell-mediated immunity following measles infection. Therefore, compounds that bind to these complement receptors, such as antibodies or other proteins, may be effective in reducing the production of IL-12 in autoimmune diseases and other conditions in which this cytokine is over-produced.
Therapeutic Applications of Interleukin-12 (IL-12)
IL-12, a cytokine that may be useful for treatment of certain diseases or for enhancing the immune response, was developed by researchers at The Wistar Institute. IL-12 is a heterodimeric protein, composed of a heavy chain (p40 subunit) and a light chain (p35 subunit) and was originally described as Natural Killer Stimulatory Factor (NKSF). Wistar is an owner of a series of U.S. and international patents on the IL-12 genes, IL-12 proteins (p35 and p40 subunits) and the uses of these compositions for treatment of a variety of conditions such as cancer and infection. Additionally, Wistar is an owner of a series of issued patents for the use of IL-12 as an adjuvant. These patents are available for license to companies developing products that incorporate IL-12.
BRAF35: The DNA-Binding Component of The BRCA2 Complex
Mutations of the human BRCA2 gene are associated with a very high risk of breast cancer, yet the majority of breast cancer patients do not carry any BRCA2 or BRCA1 mutations. Additionally the function of BRCA2 and its exact role in cell growth regulation have not yet been determined.
Wistar researches have determined that BRCA2 is part of a multiprotein complex and have identified a new protein, BRAF35 (BRCA2-Associated-Factor 35) that is the DNA-binding component of this complex. Similar to BRCA2, the expression of BRAF35 RNA is up-regulated in proliferating tissues. The BRCA2/BRAF35 complex is found associated with chromatin at the early stages of chromosome condensation. These findings suggest that the BRCA2/BRAF35 complex may have a key role in both DNA repair and cell cycle regulation. Thus BRAF35 and the BRCA2/BRAF35 complex may be useful targets for development of novel cancer therapeutics.
Novel Mitotic Checkpoint Gene
Wistar researchers have identified a new mitotic checkpoint gene, chfr, that is expressed in normal tissues but is either absent or mutated in many cancer cell lines. When dividing cells that express the wild-type chfr are exposed to nocodazole, which inhibits microtubule formation, they become arrested at prophase, while cells that lack a functional chfr gene proceed through the cell cycle and division.
The restoration of normal chfr function may be a useful target for cancer therapy. Loss of chfr function in tumor cells may be indicative of cancers that are sensitive to chemotherapy.