(58d) Pro-Apoptotic Stapled Peptides Discovered Using Bacterial Cell Surface Display Demonstrate Improved Affinity, Specificity, and Efficacy

There is currently a wealth of disease-associated proteins that are “undruggable” by modern therapeutic formats. These targets reside inside the cell, where they are inaccessible to large biologics, but lack sites where a small molecule drug could bind. Peptide therapeutics, comprised of a short chain of amino acids, are an emerging therapeutic framework that attempt to fill this gap. These peptides are small enough to enter cells while being large enough to bind and inhibit protein-protein interactions. Unfortunately, peptide therapeutics for intracellular targets have had limited success in the clinic arising from several issues including low target affinity and low proteolytic stability. To overcome these challenges, a “stapled” peptide can be formed by covalently linking two amino acids, locking the peptide into its alpha-helical state. This single modification can improve target affinity and enhance proteolytic stability. However, a key challenge in the discovery of stapled peptide therapeutics is the reliance on solid-phase synthesis, where peptides are limited to low or moderate throughput and evaluation, which greatly limits the number of peptides that can be analyzed.

In this work, we employ a novel technique, developed in the Thurber Lab, known as Stabilized Peptide Engineering by E. coli Display (SPEED) to rapidly characterize and develop >10⁹ unique stapled peptide therapeutics against members of the Bcl-2 protein family, which are important regulators of apoptosis and play key roles in resistance to chemotherapy. In this work, libraries of DNA encoding unique peptides are computationally designed by mining genome and linear peptide library data, transformed into bacteria, and presented as peptides on the cell surface. To enable characterization of stapled peptides, azide containing residues are incorporated in place of methionine through use of methionine auxotrophic bacteria. Then, copper catalyzed click chemistry (CuAAC) is used to form stapled peptides directly on the cell surface through reaction with bisalkyne staples. Finally, cells are sorted for given molecular properties by incubating with fluorescently labeled target protein. This technique was previously applied to discover an inhibitor with eightfold improved affinity against mdm2, an important oncoprotein in the p53 pathway.

Here, we improve on this technique and further demonstrate its utility in discovering potent stapled peptide inhibitors by applying it to the Bcl-2 protein family, which is comprised of five highly homologous protein targets. Because of their homology, methods that can simultaneously optimize specificity and affinity are greatly needed. First, we utilized on-cell staple scanning to identify locations for the linker that maintain target binding. The next improvement in the technique is incorporating Next Generation Sequencing (NGS) into the workflow, which enables quantitative measurement of affinity and specificity based on high-throughput fluorescent-activated cell sorting data. To identify lead molecules, computational models were constructed based on affinity and specificity profiles of >10⁵ peptides from cell sorting. Interestingly, these models generated peptides with improved specificity and affinity compared to those that emerged directly from the sorting campaign. Finally, we translated these molecules off the cell surface and evaluated their mechanisms of action and efficacy, proving they demonstrate improved efficacy in vitro over their linear counterparts. The ability to improve affinity, specificity, stability, and in vitro efficacy demonstrates SPEED’s multifaceted capability.

In summary, we apply SPEED to design tight binding, highly specific, and protease resistant stapled peptide inhibitors of Bcl-2 proteins. To our knowledge, this work represents the first high-throughput study of stapled peptide Bcl-2 inhibitors. Peptides with these improved drug-like properties promise better selective treatment for cancer with in vitro and in vivo validation of efficacy. This approach provides a framework to discover potent stapled peptide inhibitors towards other important protein targets.