Developing an in vivo platform to assess inhibitors of protein-protein interactions

Background:
In this project we will focus on developing biological tools to identify reagents capable of blocking protein-protein interactions (PPIs). PPIs are key in regulating cellular function yet current methods of identifying inhibitors and the repertoire of reagents available is limited.

Objectives:
Develop an in vivo platform for identifying inhibitors of PPIs:
1. Develop screening assay using split b lactamase: using molecular biology and protein engineering a periplasmic-based split beta lactamase enzyme screen (similar to an assay developed in the SER/DJB labs - Nature Chem Biol (2015)) will be developed. Each segment of the split beta lactamase will be fused to individual protein domains that are known to interact, such as Ras/SOScat, and Bcl-xl/BH3 domain proteins. Any inhibitor will cause cell death in the presence of antibiotic. Initially Affimers (isolated by other PhD students in DT lab) and compounds known to inhibit the PPIs will be used to test the efficacy of this assay, and to determine the assays ability in screening large libraries of individual compounds.

2. Develop a screening assay using split barnase system: an assay that results in cell survival, after inhibition of the PPI, will also be developed using a split bacterial barnase system. Expression of barnase kills E.coli cells, hence inhibition of the PPI (and barnase activity) will result in cell survival. This assay would be useful in screening large libraries of reagents as pools, such as Affimers, as only Affimers that inhibit the PPI would be isolated from the screen.


Novelty/Timeliness:
With the growing diversity of small molecules and the increasing use of biologics the need for high-throughput screening modalities is ever increasing.
This project will develop tools for primary and/or secondary screening of compounds and biologics.
Experimental Approach:
We will utilize molecular biology, protein engineering and cell biology, offering the student training in a wide range of techniques. All methods are up and running, enabling the student to make rapid progress.