Day 14 - Fragment Screening at Diamond

It's day 14 of the Diamond Industrial Liaison advent calendar. We have told about most of the techniques here at Diamond, but there is a new technique which is part of our MX village called Fragment Screening that deserves a special mention.

In the past two decades, fragment based drug discovery has emerged as a powerful method for the pharmaceutical industry to discover and develop new drugs. Such strategy relies on the identification of low molecular weight compounds that bind weakly to a target. The structural information gained on fragment-target complexes provides an excellent starting point for the development of potent and selective drugs.

Most screening efforts rely on a cascade of sensitive biophysical methods to isolate weakly binding compounds. X-ray crystallography is also an excellent, and validated, fragment screening option. The method allows screening of all potential binding locations, including allosteric sites, at once while providing a direct structural feedback on hit-target complexes. However, screening a full library of compounds via crystallography is usually laborious, resource hungry and time consuming.

I04-1 End Station

Diamond Light Source, in collaboration with the Structure Genomics Consortium, has developed a streamlined X-ray fragment screening platform. To facilitate and parallelise soaking, compounds are transferred, through an acoustic liquid dispenser, directly to crystals grown in high throughput crystallisation plates. A motorised stage located under a microscope, and associated to a touch screen user interface, significantly speeds up the crystal harvesting step. Samples are then transferred to I04-1, a fully automated MX beamline, for rapid and unattended X-ray data collection. Benefitting from immediate access to the Diamond data processing pipelines, electron density maps can be readily analysed for the presence of bound ligands.

By seamlessly integrating each step of the process, Diamond has tremendously accelerated the X-ray fragment screening approach, while reducing the experimental overheads. Hundreds of compounds are now routinely screened in a matter of days, with a rapid turnover between projects.

Crystallographic fragment screening is a powerful process to determine low molecular weight compounds binding weakly to a drug target (from um to mM Kd). The method provides direct structural information about the ligand-target interactions, at atomic level, on which chemical chemists can rely on to develop new/potential potent leads.

What can it tell me?

• Discriminate positive hits, i.e. compound binding to the target;  
• Determine molecular interaction between hits and the target;  
• Highlight conformational changes of the target which would be intractable with other methods;  
• Presence of new binding site(s) and allosteric site(s).

What does the data looks like?

• Raw data are X-ray diffraction images from the different samples;  
• Processed data provides electron density map and atom coordinates for the model of the target; 
• Positive difference density peaks might highlight a bound ligand (and or some conformational changes of the target); 
• Majority of case often difference map look like “apo” form (i.e. no compound is bound). Typical hit 1-10%, comparable with other biophysical screening methods.

Experiment requirements

• A robust and reproducible crystal system; 
• A crystal system diffracting to <2.5A, otherwise the ligand’s orientation would be too difficult to determine;
• Crystals produced in SwissCi 3 drops HTX crystallisation tray format (more formats to come);  
• The PDB model(s) and cell parameters related to the crystal system;  
• For external libraries, high compound concentration (>100 mM in DMSO, in plate format compatible with the Echo 550)

Benefits

• Small libraries of compounds provide comprehensive screening options;  
• Rapid turnover between projects;  
• Direct structural feedback about the molecular interactions between the hit and the target;  
• Rapid sample generation; 
• Fully automated data collection and data processing.

Short film on fragment screening facility available on BBC Oxford website

Coverage of the announcement of the opening of the new fragment screening facility at Diamond features in a short film on the BBC Oxford website and the ITV Meridian news website along with print articles. 

The BBC short film (~2 minutes) includes John Barker, Evotec's Vice President of Structural Biology, discussing how the new platform has helped Evotec with their projects.

The ITV news film includes interviews with Diamond CEO Prof. Andrew Harrison and Director of Life Science Prof. Dave Stuart describing the importance of structural biology at synchrotrons in the design and development of drugs.