Development of a novel high throughput screen for cytoplasmic entry of cell penetrating peptides. (360G-Wellcome-090145_Z_09_Z)

In our opinion, none of the current methods for assessing uptake of proteins by CPPs are able to give a rapid, accurate, and quantitative measure of cytosolic uptake. Our aims are therefore: (i) Generation of a high throughput, quantitative assay for the transport of proteins into the cytoplasm of mammalian cells by cell penetrating constructs. Our proposed assay is based on monitoring the cytoplasmic cleavage of a GFP-derived FRET construct We assume that GFP, which does not adhere to nor penetrate mammalian cells, will serve as a good model for proteins as cargo for penetrating constructs. In particular, we will be focusing on viral entry peptide sequences as a basis for penetrating construct design. (ii) Identification of peptide sequences that promote facile release of the GFP construct from endosomes following endocytosis. We hope to identify general sequence motifs that will promote and assist with endosomal release of proteins. (iii) Detailed FRET study of the cell entry of bromomaleimide-derived CPP bioconjugates both at cellular and < ingle molecule resolution. (iv) In addition to the screening of CPPs to enable protein cargo delivery, we will also consider other penetrating constructs. Neocarzinostatin (NCS) is known to gain cell entry in a proficient manner and ultimately interact with nuclear DNA (Schaus et al. 2001 ). It is unclear, however, what the mechanism of NCS cell entry is and whether it is ever exposed to the cyloplasm. Our proposed methodology would provide an ideal platform on which to determine this. (v) Comparison of the cell surface stability of both bromomaleimide- and disulfide-derived FRET constructs. These experiments will give an insight into whether PDis or thioredoxins could inhibit the cell entry of disulfide-derived conjugates and whether bromomaleimide-linked conjugates offer an adv2ntage in this regard. (vi) Appliccation of optimised CPPs to the cell delivery of academically and/or therapeutically interesting protein targets. This research may be performed in collaboration with interested research groups or organisations.