Analysis of Loops that Mediate Protein–Protein Interactions and Translation into Submicromolar Inhibitors

Abstract

Effective strategies for mimicking α-helix and β-strand epitopes have been developed, producing valuable inhibitors for some classes of protein-protein interactions (PPIs). However, there are no general strategies for translating loop epitopes into useful PPI inhibitors. In this work, we use the LoopFinder program to identify diverse sets of "hot loops," which are loop epitopes that mediate PPIs. These include loops that are well-suited to mimicry with macrocyclic compounds, and loops that are most similar to variable loops on antibodies and ankyrin repeat proteins. We present data-driven criteria for scoring loop-mediated PPIs, uncovering a trove of potentially druggable interactions. We also use unbiased clustering to identify common structures among the hot loops. To translate these insights into real-world inhibitors, we describe a robust, diversity-oriented strategy for the rapid production and evaluation of cyclized loops. This method is applied to a computationally identified loop in the PPI between stonin2 and Eps15, producing submicromolar inhibitors. The most potent inhibitor is well-structured in water and successfully mimics the native epitope. Overall, these computational and experimental strategies provide new opportunities to design inhibitors for an otherwise intractable set of PPIs.

Emerging Methods and Design Principles for Cell‐Penetrant Peptides

“…[64][65][66]137] Thed eterminants for passive penetration of small cyclic peptides are relatively clearly defined, but there remain several open questions.C an this chemical space be successfully mined for lead compounds,e specially inhibitors of intracellular protein-protein interactions?C an backboneshielding networks of internal hydrogen bonds be designed de novo,e specially for peptides that organize ad esired loop structure? [138][139][140] How extensively can polar side chains be incorporated into these structured macrocycles while maintaining passive penetration? [70,136,141,142] Can these peptides be engineered as substrates for endogenous peptide transporters?…”

Understanding and designing head‐to‐tail cyclic peptides

“…For example, several methods have been developed to identify crucial binding regions in protein–protein interactions. These include the identification of “hot segments” using Rosetta Peptiderive, “hot loops” using LoopFinder, helical interfaces using HippDB, and β‐sheets at interfaces . Additionally, PepComposer developed a methodology to design peptides given a target structure and binding site .…”

Targeting intracellular protein–protein interactions with cell-permeable cyclic peptides

“…Further SAR studies yielded a highly-potent, cell-permeable cyclic inhibitor with a K i of 4.7 nM against the MLL1-menin interaction (Figure 2). More recently, the stapling strategy has been applied to generate structurally constrained hairpin loops as PPI inhibitors [18]. These examples demonstrate that naturally occurring epitope sequences at PPI interfaces, when available, can serve as good starting points for rational design of potent cyclic peptide PPI inhibitors.…”