000223452 001__ 223452
000223452 005__ 20190317000602.0
000223452 0247_ $$2doi$$a10.5075/epfl-thesis-7293
000223452 02470 $$2urn$$aurn:nbn:ch:bel-epfl-thesis7293-9
000223452 02471 $$2nebis$$a10788149
000223452 037__ $$aTHESIS
000223452 041__ $$aeng
000223452 088__ $$a7293
000223452 245__ $$aScreening of structurally diverse bicyclic peptide libraries by phage display
000223452 269__ $$a2016
000223452 260__ $$aLausanne$$bEPFL$$c2016
000223452 300__ $$a123
000223452 336__ $$aTheses
000223452 500__ $$aLa date de soutenance 14 décembre 2016, mentionnée sur la page de titre, est erronée
000223452 502__ $$aDr Anne-Sophie Chauvin (présidente) ; Prof. Christian Heinis (directeur de thèse) ; Dr Ruud Hovius, Prof. Ali Tavassoli, Prof. Tom N. Grossmann (rapporteurs)
000223452 520__ $$aBicyclic 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.
000223452 6531_ $$aPeptide
000223452 6531_ $$abicyclic peptide
000223452 6531_ $$amacrocycle
000223452 6531_ $$ain vitro evolution
000223452 6531_ $$aphage display
000223452 6531_ $$ab-catenin
000223452 6531_ $$aICAT
000223452 6531_ $$aactin
000223452 6531_ $$athymosin beta-4
000223452 700__ $$0246702$$aBertoldo, Davide$$g229605
000223452 720_2 $$0241317$$aHeinis, Christian$$edir.$$g163670
000223452 8564_ $$s15214376$$uhttps://infoscience.epfl.ch/record/223452/files/EPFL_TH7293.pdf$$yn/a$$zn/a
000223452 909C0 $$0252109$$pLPPT$$xU11959
000223452 909CO $$ooai:infoscience.tind.io:223452$$pthesis-bn2018$$pDOI$$pSB$$pthesis$$qDOI2$$qGLOBAL_SET
000223452 917Z8 $$x108898
000223452 917Z8 $$x108898
000223452 917Z8 $$x108898
000223452 917Z8 $$x108898
000223452 918__ $$aSB$$cISIC$$dEDCH
000223452 919__ $$aLPPT
000223452 920__ $$a2016-12-9$$b2016
000223452 970__ $$a7293/THESES
000223452 973__ $$aEPFL$$sPUBLISHED
000223452 980__ $$aTHESIS