BBBRC EASTBIO DTP - Monobody biosimilars as tools to probe the differential dynamic regulation of the EGFR network by protein-protein interaction switches

Extracellular signalling helps control and guide intercellular processes. A problem therefore arises when the network reading these signals becomes broken or locked in an undesirable state. The epidermal growth factor (EGFR) network is one such network, frequently altered in many human diseases including, neurodegeneration, chronic kidney disease and cancers. This well studied, but still poorly understood network is known to adopt many possible states, driven by molecular ‘switches’, altering the state of EGFR driven survival, proliferation and differentiation. Historically, small molecule probes are poor choices for systematic protein-protein interaction inhibition. We propose a project which will allow the student to systematically tackle and perturb the EGFR network at key locations. As a member of the European FP7 project PRIMES (Protein interaction machines in oncogenic EGF receptor signalling), the Auer lab has access to unpublished data intricately describing the EGFR network in terms of protein-protein interactions involved in cell signalling. Using >100 bait proteins, >400 bait-prey interactions were identified, resolving differentially up and down regulated edges (PPIs). Using several criteria, including observed protein expression levels and interaction strength changes in perturbed vs non-perturbed signalling, we will identify high affinity, high specificity, small protein domain (biosimilar) modulators, and apply them for further improving the understanding of EGFR network regulation and information flow. The project comprises the following work packages; (1) to prioritise key information flow protein targets for mammalian expression based on currently proprietary knowledge on EGFR signalling. (2) Development of a second generation CTB monobody library. The Auer lab routinely makes use of phage display technologies to identify peptidic and biosimilar starting points. Of particular interest are monobodies, termed ‘antibody mimetics’. Their small size (~10 kDa) improves their pharmokinetic profile and their lack of disulphide bonds allows their use against intracellular targets in reducing as well as oxidizing environments for localization and disruption studies [2]. (3) Application of the Phage-CONA screening technique to identify novel monobody inhibitors of EGFR-relevant protein-protein interactions nodes. Phage-CONA is a novel phage display variant, that combines phage display with the CONA (Confocal Nanoscanning) method [3]. In Phage-CONA the target protein and the monobody hits are labelled with different fluorescent proteins for ratiometric imaging. Only the highest affinity binders will be taken forwards to sequencing and further analysis. (4) To study the biophysical characteristics of monobody inhibition using single molecule spectroscopic techniques: Single-molecule techniques overcome inherent limitations of ‘bulk’ fluorescence measurements by enabling the measurement of the distribution of states within a system, monitoring binding as a function of time. Specifically, the student will use “time-correlated single photon counting” (TCSPC) combined with MPFD (Multi-Parameter Fluorescence Detection) to reveal the mechanism of monobody target binding. (5) To functionally evaluate the monobody inhibitors in intact cell systems: The student will have access to a large range of cellular phenotypic assays developed in the Carragher lab for evaluation of monobody effects. These include image-based multiparametric high content cell painting assays across IPSC-derived neuronal and cancer cell line panels to classify mechanism-of- action and perform pharmacogenomic and pharmacoproteomic analysis to identify biomarkers and patient stratification hypothesis, live-cell kinetic apoptosis assays, senescence bypass assays, autophagy assays, 3D colony formation, neurite outgrowth, stem cell renewal, live cell kinetic 2D scratch wound migration assays, and tissue fibrosis assays to evaluate and compare functional activity across pathophysiological bioassays representing multiple disease areas.

The outcome of the proposed PhD work will be (a) series of novel tool reagents to study EGFR signalling by modulation of so far undrugable network nodes, and (b) increased knowledge of dynamic EGFR network regulation in cell physiology.