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Actin is the most abundant protein in eukaryotic cells and is key to many cellular functions. The filamentous form of actin (F-actin) can be studied with help of natural products that specifically recognize it, as for example fluorophore-labeled probes of the bicyclic peptide phalloidin, but no synthetic probes exist for the monomeric form of actin (G-actin). Herein, we have panned a phage display library consisting of more than 10 billion bicyclic peptides against G-actin and isolated binders with low nanomolar affinity and greater than 1000-fold selectivity over F-actin. Sequence analysis revealed a strong similarity to a region of thymosin-β4, a protein that weakly binds G-actin, and competition binding experiments confirmed a common binding region at the cleft between actin subdomains 1 and 3. Together with F-actin-specific peptides that we also isolated, we evaluated the G-actin peptides as probes in pull-down, imaging, and competition binding experiments. While the F-actin peptides were applied successfully for capturing actin in cell lysates and for imaging, the G-actin peptides did not bind in the cellular context, most likely due to competition with thymosin-β4 or related endogenous proteins for the same binding site.

Macrocycles provide an attractive modality for drug development, but generating ligands for new targets is hampered by the limited availability of large macrocycle libraries. We have established a solution-phase macrocycle synthesis strategy in which three building blocks are coupled sequentially in efficient alkylation reactions that eliminate the need for product purification. We demonstrate the power of the approach by combinatorially reacting 15 bromoacetamide-activated tripeptides, 42 amines, and 6 bis-electrophile cyclization linkers to generate a 3780-compound library with minimal effort. Screening against thrombin yielded a potent and selective inhibitor (Ki = 4.2 ± 0.8 nM) that efficiently blocked blood coagulation in human plasma. Structure–activity relationship and X-ray crystallography analysis revealed that two of the three building blocks acted synergistically and underscored the importance of combinatorial screening in macrocycle development. The three-component library synthesis approach is general and offers a promising avenue to generate macrocycle ligands to other targets., Combination of three efficient chemical reactions allows for solution-phase synthesis of 3780 macrocycles and identification of potent thrombin inhibitor.