Design of novel BH3-like sequences and their application for diagnosisof anti-apoptotic blocks in cancer cells

• Applicant: Dr. Raheleh Rezaei Araghi
• Subject Area: Biological and Biomimetic Chemistry
• Term: from 2012 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 225104808

Anti-apoptotic and pro-apoptotic Bcl-2 proteins are necessary for regulating cell death program. Anti-apoptotic receptors can inhibit apoptosis by binding to the ý-helical BH3 region of the pro-apoptotic proteins by means of their conserved hydrophobic groove. Overexpression of Bcl-2 anti-apoptotic members occurs in diseases such as cancer, therefore, the design of suitably modified helical BH3 peptides with increased affinity and selectivity holds promise for new avenues of cancer therapy. Helix-stabilizing strategies for BH3 inhibitors that bind their receptors in an ý-helical conformation can increase protein binding by virtue of conformational preorganization. However, the virtually unlimited structural variations that can be studied, presents an extreme challenge with respect to optimizing the location and type of helix-stabilizing elements. Therefore, an innovative approach is proposed to apply structural and chemical modification in split-and-pool libraries in order to prepare and screen a large number of cross-linked and non-cross-linked redesigned peptides. This approach has a great potential to overcome many of the difficulties by slow, costly, and empirical methods used in modifying ý-helical peptides today. Olefinic dialkylated amino acids will be incorporated into peptides during chain extension, followed by closure of the macrocyclic bridge using ruthenium-mediated ring-closing metathesis on a solid support. The split-and-pool libraries are suitable for both on-bead modifications and on-bead screening against fluorescently labelled anti-apoptotic proteins without purification. The detailed in vitro characterization of high-affinity modified BH3 peptides will be carried out in solution, including investigation of their helical content, binding affinity, binding kinetics, and affinity quantification. Next, as the optimum goal of the project, the panel of high-affinity modified BH3 peptides will be used to identify anti-apoptotic receptors that are responsible for cell survival. This is performed by „BH3 profiling“, which measures the cytochrome c release of mitochondria isolated from tumor cells. Applying this assay to the set of highly selective BH3 variants isolated from the above screen, would enable the identification of chemo-resistant anti-apoptotic receptors, which have been difficult to differentiate from other similar receptors.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Dr. Amy Keating