In Vitro-Evolved Peptides Bind Monomeric Actin and Mimic Actin-Binding Protein Thymosin-β4

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.

Screening of structurally diverse bicyclic peptide libraries by phage display

Davide Bertoldo

Bicyclic peptides are an attractive molecule format for the development of therapeutics and research tools. Our laboratory is specialized on the development of bicyclic peptide ligands by phage display. In brief, libraries of randompeptides each containing three cysteines are displayed on the surface of a phage and cyclized with a trivalent thiol-reactive chemical reagent. Ligands of a target of interest are isolated by affinity selection and their sequence identified by DNA sequencing. The first goal of my thesis was to test if more diverse bicyclic peptide libraries can be generated if linear peptides on phage are cyclized in parallel with multiple different chemical cyclization linkers. Towards this end, we cyclized peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) with three different chemicals and panned the library against the protease urokinase-type plasminogen activator (uPA). Interestingly, different peptide sequences were enriched when the phage peptide library was cyclized with the different chemical linkers, indicating that a larger diversity of bicyclic peptides is generated. Screening the larger library generated binders with a higher binding affinity. In the second project I carried out, I isolated bicyclic peptide ligands to a protein of therapeutic interest, b-catenin. b-catenin is the main translational co-activator of theWnt cell signaling pathway. Pathological upregulation of b-catenin is associated with the majority of colorectal cancer. Blocking b-catenin activity is thus an attractive therapeutic approach. However, so far the development of b-catenin inhibitors has been challenging due to the flat and featureless surface of the protein, lacking binding pockets for synthetic ligands. Screening a phage display library comprising >12 billion bicyclic peptides yielded binders for different protein epitopes. The binding site of four peptides was identified to correspond to that of ICAT (inhibitor of b-catenin and Tcf), which is a prime target site on b-catenin for therapeutic intervention and to which no synthetic ligands could be isolated so far. In the third project, we isolated and characterized bicyclic peptides binding to globular actin (G-actin). G-actin constitutes the fundamental building block of microfilaments (also named F-actin) in eukaryotic cells. Beyond this structural function, G-actin has a role in gene transcription and chromatin remodeling. While specific F-actin probes are available for in vitro applications, G-actin probes are lacking. To fulfil this demand, we screened a library of bicyclic peptides for G-actin binders and isolated ligands with dissociation constants in the low nanomolar range. The isolated peptides were found to bind to the same surface region as thymosin b4 and a range or marine toxins. The bicyclic peptide G-actin ligands allowed measurement of the binding affinity of natural G-actin ligands in fluorescence anisotropy competition assays.

EPFL2016

Screening of structurally diverse bicyclic peptide libraries by phage display

Davide Bertoldo

EPFL2016