Abstract C212: Discovery of high affinity cyclic peptidomimetics binding Mcl-1 and Ras.
Authors: Josephson Kristopher , Ma Zhong , Wang Zhaolin , Sun Yili , Tobe Sylvia , Perlmutter Sarah , Vyasamneni Rohit , Ye Ping , Boyer Nicolas , Arata Micelle , Pattavina Kelli , Seyb Kathleen , Zheng Hong , Sollomoni Imelda , Nims Ezekiel , de Koning Emily , Ricardo Alonso , Treco Douglas 
Published: August 2014

Abstract
Many well-validated oncology targets regulate signaling pathways through interactions with other proteins. Addressing these targets, which often lack suitable small molecule binding sites, requires larger and more complex molecules that are capable of binding relatively large or flat surfaces. We are testing whether this target class can be addressed by using in vitro selection to screen extremely large cyclic peptidomimetic libraries with novel design features.
Peptide-mRNA display libraries comprised of approximately 1 trillion cyclic peptidomimetics were created by in vitro translation using a mixture of natural, unnatural, and N-methylated amino acids. Pooled libraries were subjected to multiple rounds of selection and amplification to identify sequences that bound with high affinity to immobilized Mcl-1 or Ras.
Selected Mcl-1 cyclic peptidomimetics were synthesized and their binding and specificity were studied by surface plasmon resonance (SPR) using immobilized Mcl-1 and Bcl-2. Fluorescence polarization (FP) was used to demonstrate that isolated peptidomimetics competed with Bim-derived peptides (IC50≈100-400 nM), a measure of their ability to block the interactions between Mcl-1 and binding partners. The co-crystal structure of one Mcl-1 inhibitor revealed that the cyclic peptidomimetic bound the BH3 helix-binding grove by forming a β-hairpin, an unprecedented type of interaction with Bcl-2 family members. The cell permeability of analogs of this compound was verified by fluorescence microscopy and by induction of apoptosis.
In a second example, a cyclic peptidomimetic selected against mutant H-Ras was shown to bind both H-Ras and K-Ras with high affinity by SPR (KD=2.5 nM) and FP (Ki≈£10 nM). Despite its high affinity, this compound did not significantly impact Ras activity in several in vitro assays (GST-Raf-RBD binding, GTPase, and GDP/GTP exchange). A co-crystal structure with mutant K-Ras, however, revealed that this compound binds a novel site in the C-terminus of the Ras G-domain. Remarkably, this compound also forms a β-hairpin, and like the Mcl-1 compound, also appears to enter cells. Experiments to test how this compound may affect Ras signaling or localization, and the function of this novel site in cells or in vitro with full-length lipid modified Ras are in progress.
The target-bound conformation of both inhibitors reveled a network of intramolecular hydrogen bonds that are predicted to reduce interactions with water and may help to explain the intrinsic cell permeability of these molecules. The use of extremely large peptidomimetic libraries built with novel chemical diversity appears to be an extremely efficient platform for isolating high affinity binders for hard-to-target intracellular targets, yielding novel binding modes and binding sites that are unlikely to be identified by typical small molecule approaches or rational design.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C212.
Citation Format: Kristopher Josephson, Zhong Ma, Zhaolin Wang, Yili Sun, Sylvia Tobe, Sarah Perlmutter, Rohit Vyasamneni, Ping Ye, Nicolas Boyer, Micelle Arata, Kelli Pattavina, Kathleen Seyb, Hong Zheng, Imelda Sollomoni, Ezekiel Nims, Emily de Koning, Alonso Ricardo, Douglas Treco. Discovery of high affinity cyclic peptidomimetics binding Mcl-1 and Ras. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C212.