JHU/CCR Fellowship in Molecular Targets and Drug Discovery Technologies Project Details

Project Sponsor/Mentor:	Charles Vinson
Title:	Principal Investigator
Address:	Room 3128 Bldg. 37
Telephone:	301-496-8753
Fax:	301-496-8419
Email:	vinsonc@mail.nih.gov
Sponsoring Laboratory/Branch:	Laboratory of Metabolism

Project Title:	Compounds that inhibit B-ZIP DNA binding
Target(s) of Interest:	Inhibiting B-ZIP DNA binding in human cancer

Project Synopsis:
We have conducted a high-throughput screen to identify small molecules that inhibit the DNA binding of B-ZIP transcription factors that include CREB, AP-1, and C/EBP proteins. These transcription factors have been implicated in many different cancers. We identified one class of compounds, arylstibonic acids, that bind to the B-ZIP domain and inhibit DNA binding. Several cell-based assays demonstrate that these compounds inhibit the DNA binding of B-ZIP in cells. However, they are benign to most cell lines and in mice suggesting the B-ZIP function is not critical for cell viability. To test this idea, we examined the pediatric cancer, clear cell sarcoma that contains a characteristic translocation that produces a chimeric proteins between the transactivation domain of EWS and the DNA binding domain of the B-ZIP protein ATF1. These cells are killed by the arylstibonic acids. The project will evaluate the mechanism of action of these compounds in this pediatric cancer to help determine the potential for clinical applications. We have conducted a high-throughput screen, in collaboration with the Developmental Therapeutics Program, to identify small molecules that inhibit the DNA binding of B-ZIP transcription factors that include CREB, AP-1, and C/EBP proteins. These transcription factors have been implicated in many different cancers but no small molecules have been identified that inhibit their function. We identified one class of compounds, arylstibonic acids, that bind to the B-ZIP domain and inhibit DNA binding. Fortunately, the Developmental Therapeutics Program has a library of 50 different arylstobinic acids which as allowed us to do a detailed structure activity relationship analysis. We have identified a compound that binds to the B-ZIP domain at 30 nM. This biochemical analysis of the specificity of action of this library of arylstibonic acids for different B-ZIP domains is ongoing. Arylstibonic acids contain an antimony element which is rarely observed in clinically used small compounds. This aspects of the chemistry makes this project particularly novel. We have used several cell-based assays to evaluate if these compounds inhibit the DNA binding of B-ZIP in cells. Transient transfection of reporter constructs indicates that the arylstibonic acids that are active in vitro also are active in cells assays while arylstibonic acids that are not active in vitro are not active in cell assays. Furthermore, reporter constructs that are dependent on DNA sequences bound by B-ZIP proteins are inhibited by arylstibonic acids while reporter constructs that are not dependent on DNA sequences bound by B-ZIP proteins are not inhibited by these compounds. The promiscuity of these compounds at inhibiting the DNA binding of different B-ZIP domains is also observed in the transient transfection reporter assays. In total, these transient transfection assays recapitulate what is observed in vitro. A second cell-based assay we have used is FRAP (Fluorescent Recovery After Photobleaching). In this assay, we made chimeric proteins containing the B-ZIP domain and GRP (Green Fluorescent Protein). These constructs are transfected in to mammalian cells resulting in a green nucleus when the cells are imaged using fluorescent microscopy. We then photobleach a small part of the nucleus and monitor the diffusion of the B-ZIP-GFP chimeric protein into the bleached part of the nucleus. This analysis indicates that the presence of the Arylstibonic acids makes the chimeric protein move more rapidly in the nucleus. Extensive controls using B-Zip mutants indicates that the increase in mobility caused by the arylstibonic acids is comparable to a B-ZIP domain that is unable to bind to DNA. Again, the promiscuousness of the arylstibonic acids at inhibiting the DNA binding of B-ZIP domains is observed in this assay. However, the DNA binding of other classes of transcription factors the not inhibited by the arylstibonic acids. The challenging aspects of these results is that the arylstibonic acids are inhibiting the DNA binding of all B-ZIP proteins but the growth of the cells is not affected suggesting that B-ZIP proteins are not critical for cell viability. To test this idea, we examined a pediatric cancer called Clear Cell Sarcoma that contains a characteristic translocation that produces a chimeric proteins between the transactivation domain of EWS and the DNA binding domain of the B-ZIP protein ATF1. In this case, we know that cell viability is dependent on the activity of a B-ZIP protein. The growth of this cell line is inhibited by the arylstibonic acids while the growth of other cells lines is not inhibited by the arylstibonic acids. Soft colony formation of the Clear Cell Sarcoma is also inhibited by the arylstibonic acids while soft colony formation of other cells is not affected by the arylstibonic acids. A particularly promising observation is that in mice, 50 uM serum levels can be obtained without any obvious detrimental affects. A previous JHU fellow did the FRAP experiments and the evaluation of the Clear Cell Sarcoma. She is presently working for Astra-Zeneca in the area. The project will evaluate the mechanism of action of arylstibonic acids at inhibiting the growth of Clear Cell Sarcoma. Recent data from the laboratory has identified an arylstibonic acid that can inhibit B-ZIP DNA binding at 30 nM but no cell based data is available. One aspect of the project will be to determine the biological properties of these newly identified arylstibonic acids.
Fellow Research Plan:
Currently, the group is doing biochemical studies that are examining a library of 50 arylstibonic acids for binding to different B-ZIP domains. Initial studies have identified a compound that binds at 30 nM to various B-ZIP domains. We are extending these studies to determine if this compound is active is cells. Previous studies has demonstrated that an arylstibonic acids that binds to various B-ZIP domains at 3 uM is active in cells and is able to stop the growth of a pediatric sarcoma, a clear cell sarcoma, that contains a chimeric protein containing a B-ZIP domain. The viability of this sarcoma is dependent on the activity of the B-ZIP domain. Two projects are available. One addresses the biochemical interaction of these compounds with the B-ZIP domain. We have initiated a collaboration of David Waugh at NCI Frederick who is doing X-ray studies to determine cocrystal studies between the arylstibonic acids and the B-ZIP domain to help gain insight into the structural interaction between these two components. These studies will be essential in the development of more active and specific arylstibonic acids. In the laboratory, the project will examine in biophysical detail the interaction between the arylstobinic acids. These studies will include circular dichroism spectroscopy, analytical ultracentrifugation and microcalorimetry. The second project will address the mechanism of action of the arylstibonic acids at inhibiting the growth of the clear cell sarcoma that contains a chimeric protein with the EWS transactivation domain and the ATF1 B-ZIP DNA binding domain. Presently, we are obtaining additional clear cell sarcoma clinical isolates to help confirm that the inhibition of growth observe for the single isolate we have is a general propertiy of this pediatric cancer. These studies will include cell cycle analysis, mRNA microarrays studies, and SiRNA to ATF1. Additional studies will be done as we learn more about these compounds. Furthermore, we are planning on doing animal studies where we produce xenographs of the cancer in mice and determine of the arylstibonic acids can inhibit growth and a more complex biological context.