Development of a Knottin Phage Display library for confocal scanning and high content imaging

Cystine-knot miniproteins, also known as Knottins are a structural family (typically 30–50 amino acids) that share a common fold, in which one disulfide bond threads through a macrocycle created by two other disulfide bonds and the peptide backbone, creating a rigid molecular ‘knot’ that confers high chemical, thermal and proteolytic stability. Knottins possess loop regions that have been shown to tolerate amino acid mutations, designating them as promising molecular scaffolds for drug discovery applications [1]. The online KNOTTIN database is a valuable resource for research updates and standard information related to knottins (http://knottin.cbs.cnrs.fr).

The project comprises 4 main parts: (A) Development of several variations of a > 1010 mer phage display library for two main applications. The first variant will be produced for screening against target proteins in-vitro using a combination of phage display and confocal scanning, Phage-CONA. (B) Phage CONA is a novel method that combines phage display with the CONA (Confocal Nanoscanning) method [2] in which the target protein is fluorescently labelled with a different excitation/emission spectrum to the one of the phage library knottin-FP (Fluorescent Protein). Adding the phage library knottin-FP selected by panning against the target protein to bead bound labelled target protein allows ratiometric measurements between the fluorescence of the two different fluorophores. This allows the creation of a quantitative score representing the relative affinity of the peptide. Only binders with the highest affinity score will be sequenced to determine their primary structure. Thus detected knottin binders will be used for intracellular localization and pathway disruption studies as well as determining the biophysical characteristics of inhibition. (C) The second variant will comprise a library of expressed and encoded knottins which will be applied for phenotypic screening in multiparametric high content “cell painting” assays, quantifying several hundreds features per cell, applied across a panel of distinct cancer cell lines, to classify phenotypic mechanism-of-action (vs annotated reference compound set) [3]. Secondary functional assays will include live-cell kinetic apoptosis, cell migration assays, patient-derived drug sensitive & resistant ovarian cancer/cell models and, live cell/kinetic, T-cell mediated tumour killing assays. (D) High affinity binders identified by Phage-CONA for known or potential novel target proteins in cancer, e.g. ubiquitin conjugating enzymes, E2s, or proteins of cell cycle regulation, e.g. Survivin, Ska, CENP32 will be evaluated for functional activity in assays used for part (C) of the project.

This cross-disciplinary project, encompassing areas of molecular and cellular biology, biochemistry, biophysics, fluorescence spectroscopy, imaging and assay technology development, will allow the PhD candidate to acquire skills in a vast repertoire of techniques. Besides classical cell and molecular techniques, including mammalian and bacterial cell culture and handling, protein expression and purification, enzymatic assays, a range of cutting-edge technologies and advanced equipment will be used, such as the high content - high speed confocal imaging on the Opera (PerkinElmer) ImageXpress (Molecular Devices) and the laser-enabled analysis and processing (LEAP) of live cells in situ, IncuCyte® live cell kinetic imaging (Essen Biosciences) and multiparametric image analysis using open-source analysis methods (e.g. Cellprofiler)