Northern Institute for Cancer Research

Structural Biology

Structural Biology

About

Structural biology and cell-free assays play a central role in prosecuting drug discovery campaigns against challenging targets.

Research Leaders

Jane Endicott
Martin Noble

Our research programme

While a target is being investigated by the Target Validation team, we investigate platforms for:

  • protein expression
  • biophysical characterisation 
  • crystallography
  • assay development

These evaluations let us identify strategies for inhibiting the target. They lay the foundation for hit-finding strategies and iterative structure-based drug design.

Protein expression

We identify fragments of the target that mediate either catalytic activity or macromolecular recognition processes (protein-DNA, protein-RNA or protein-protein interaction). We do this based on functionally annotated databases and the literature. 

We encourage heterologous expression of target proteins, in bacterial or insect cells. This allows us to generate sufficient quantities to use in our reagent-intensive activities. 

Guided by precedent where available, we typically prepare tens of different expression constructs. We find those which are most suitable for use in assay or structural biology. 

We have adapted high-throughput techniques to allow parallel E.coli expression screening of up to 24 constructs. We also use baculoviral expression and co-expression strategies to produce target proteins in insect cells.

Biophysical assays and crystallography

We use many strategies for identifying hit matter against a target.  Increasingly, we apply fragment screening technologies to novel targets. 

We use Echo® Acoustic Liquid Dispensing Technology to dispense volumes as low as 2nl into assay wells or crystal-containing droplets. We use orthogonal assays to verify hit matter.

Homogenous assays are those where all the constituents are in a solution phase. We've used a PHERAstar® plate reader to build homogenous assays. We've based this on:

  • time-resolved fluorescence
  • fluorescence polarization
  • conventional spectroscopy

To determine the affinity of protein:ligand interactions we use surface plasmon resonance. We use the latest GE Life Sciences Biacore S200 Instrumentation. 

Using this, we can screen hundreds of inhibitors against a target in a matter of hours. This also lets us interrogate association and dissociation rate constants.

Hit validation

We validate our initial hits using a series of orthogonal techniques. These include:

  •  Isothermal Titration Calorimetry
  • Nuclear Magnetic Resonance
  • Differential Scanning Fluorimetry

These techniques give us a greater understanding of how the small molecule fragments interact with our target protein. 

They allow us to assess the binding affinity and the stabilization of the protein:fragment complex.

Perhaps the most sensitive and robust technique for detecting fragments that bind to a target is crystallographic fragment screening. The preparation of high quality crystals allows structures to be determined for molecules that have millimolar affinity for their target. This can be a useful starting point for iterative structure-based drug design.

We use structure-based drug design where we can determine atomic resolution three-dimensional structures for a hit bound to our targets. This rapidly improves its target-affinity and pharmacological properties. 

The process is an iterative one. Each cycle involving stages of design, synthesis and characterisation.