ABSTRACT
Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.
Subject(s)
Macrocyclic Compounds , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/chemical synthesis , Humans , Cell Membrane Permeability , Peptides, Cyclic/chemistry , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/metabolism , Molecular Structure , Small Molecule Libraries/chemistry , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/pharmacology , Small Molecule Libraries/metabolism , Thrombin/metabolism , Thrombin/antagonists & inhibitors , Thrombin/chemistry , Crystallography, X-Ray , Sulfhydryl Compounds/chemistry , Models, MolecularABSTRACT
Thiol groups are suitable handles for site-selectively modifying, immobilizing or cyclizing individual peptides or entire peptide libraries. A limiting step in producing the thiol-functionalized peptides is the chromatographic purification, which is particularly laborious and costly if many peptides or even large libraries are to be produced. Herein, we present a strategy in which thiol-functionalized peptides are obtained in >90% purity and free of reducing agent, without a single chromatographic purification step. In brief, peptides are synthesized on a solid support linked via a disulfide bridge, the side-chain protecting groups are eliminated and washed away while the peptides remain on resin, and rather pure peptides are released from the solid support by reductive cleavage of the disulfide linker. Application of a volatile reducing agent, 1,4-butanedithiol (BDT), enabled removal of the agent by evaporation. We demonstrate that the approach is suited for the parallel synthesis of many peptides and that peptides containing a second thiol group can directly be cyclized by bis-electrophilic alkylating reagents for producing libraries of cyclic peptides.
Subject(s)
Disulfides , Solid-Phase Synthesis Techniques , Peptides/chemistry , Peptides, Cyclic , Reducing Agents , Solid-Phase Synthesis Techniques/methods , Sulfhydryl Compounds/chemistryABSTRACT
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-ß4, 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-ß4 or related endogenous proteins for the same binding site.
Subject(s)
Actins/chemistry , Microfilament Proteins/chemistry , Peptides, Cyclic/chemistry , Thymosin/chemistry , Actin Cytoskeleton/chemistry , Binding Sites , Binding, Competitive , HeLa Cells , Humans , In Vitro Techniques , Marine Toxins/chemistry , Oxazoles/chemistry , Peptide Library , Protein Binding , Protein Conformation , Structure-Activity RelationshipABSTRACT
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 (K i = 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.
