The aim of this project, initiated in November 1999, is to select specific peptide ligands against the chemokine coreceptors CCR5/CXCR4 used by HIV-1 to infect target cells. These peptide ligands should help in (a) the design of novel peptidic inhibitors of the HIV-1 infection and (b) the characterisation of the interaction which takes place between the chemokine receptor and clinically-relevant ligands. During this project, we plan to
(1) generate novel and diverse phage-peptide librariesIn vitro evolution by exon shuffling1 has been used by combining site-specific recombination (Cre/lox system) with self-splicing events (group I intron) for the generation of a large and diverse peptide library (˜ 1010) displayed on filamentous bacteriophage. This peptide library has been characterised by sequencing 20 clones obtained after the recombination.In collaboration with the University of Geneva (Dr. O. Hartley, Faculty of Medicine), novel eukaryotic cell lines expressing different levels of both chemokine coreceptors have been established.With these tools in place, we are now ready to begin the selection procedures for the isolation of specific anti-CCR5 peptide ligands using various selection procedures:
(2) to characterise stable cell line expressing different levels of the chemokine coreceptors on the cell surface
(3) establish novel cell surface selection procedure to isolate specific and high affinity phage-peptide ligands against the coreceptors
(1) High affinity peptide ligands eluted with low pHFisch, I., Kontermann, R.E., Finnern, R., Hartley, O., Solergonzalez, A.S., Griffiths, A.D. & Winter, G. A Strategy Of Exon Shuffling For Making Large Peptide Repertoires Displayed On Filamentous Bacteriophage. Proc. Natl. Acad. Sci. USA 93, 7761-7766 (1996).
(2) High specificity and high affinity peptide ligands eluted by equilibrium displacement using the natural chemokine
(3) Internalised specific peptide ligands eluted after the induction of cellular sequestration of receptor bound phage-peptide.