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XChem: X-ray structure-accellerated, synthesis-aligned fragment medicinal chemistry
Fragment-based screening is now well-established as a powerful approach to early drug ("lead") discovery. Of the many suitable biophysical techniques, X-ray crystallography was one of the first to be used, and is the most directly informative (reference); however, the experimental overheads have historically been too high for it to be widely used for primary screening.
At Diamond beamline I04-1, the full X-ray screening experiment has now been implemented as a highly streamlined process, allowing up to 500 crystals to be soaked and harvested in a day, and collected in 24 hours beamtime. The process covers soaking, harvesting, automatic data collection, and data analysis; and fragment libraries are available, or users can bring their own.
Since April 2015, the XChem facility has been available to users with dedicated weekly beamtime, and is now part of the MX User Programme (apply here); as a world-wide first facility, we welcome international proposals. The future focus is on exploring how best to harness the technique to proceed rapidly to potent compounds.
The facility is based at beamline I04-1 and nearby Lab 36, where the soaking and harvesting is performed.
In practice, the experiment will span a few days and even multiple visits to establish crystals' suitability. Users must generate the crystals in their home lab, and are required to come and perform soaking and harvesting themselves: multi-day Lab Visits will be scheduled separate from your normal beamtime allocation. In contrast, users do not need to be present for the X-ray data collection, which will also not be booked to your usual BAG or other allocation. A local contact will be assigned, same as for beamtime.
The workflow of an ideal experiment is outlined in the adjacent schema. In practice, the first three steps are iterative and require a few dozen crystals, and in difficult cases even several Lab Visits; but associated diffraction testing will be fitted in during the Lab Visit where possible. The final "Full run" soaking and harvesting will be scheduled once the soaking protocol is confirmed (in favourable cases during the same Lab Visit).
Data analysis builds on the existing automatic data processing, and we have developed tools to streamline density interpretation and refinement (PanDDA and XChemExplorer). Use of these tools at Diamond is optional but highly recommended: they are taylored very specifically to the many-dataset problem, and have been extensively tested. In future, we will also support analysis and presentation of hit results, and depositing hit structures.
Full details are provided here.
Academic users are required to publish their results and (eventually) deposit their hit structures in the PDB, as for all MX beamtime. In addition, we will occasionally request brief reports on how hits are progressing to potent compounds, to help us evaluate the effectiveness of the facility.
Access is collaborative, as it involves extensive staff support, experiment customization and significant beamtime; therefore Diamond requires us to request co-authorship on papers relying on the results (usually: the PBS and assigned Local Contact).
Projects from industry can also have "academic" status, provided there is a pledge to publish and deposit the whole story, including the follow-up chemistry and characterisation. This should be spelled out when applying.
Cocktail- vs singleton soaking
Historically, because of the overhead of X-ray fragment screening, crystals were soaked with cocktails of 4-10 compounds. Our process makes it realistic to do singleton soaking (one compound per crystal), allowing a higher effective concentration per soak; this is what we currently recommend (under evaluation).
However, libraries prepared as cocktails are accommodated perfectly well; and we also support on-the-fly cocktails, i.e. multiple compounds dispensed into one crystal drop (under evaluation).
We provide fragment libraries (below), but users are welcome to bring their own: they merely need to be in ECHO-compatible plates. (We do not need to know what's in them, and we provide a template COSSH.)
Compound concentration and solvents
The aim is to maximise the effective compound concentration seen by the crystal; also, not all solvents are compatible with a crystal (e.g. DMSO often binds to binding sites). Thus, we advocate the following:
Dispensing from non-DMSO is possible, but still being optimised.
Crystal harvesting is manual but robot-assisted (i.e. not fully automatated): this enables fast enough mounting (the record is 200 crystals/hour) that this is no longer a bottleneck.
An ideal system yields crystals that grow easily in many drops, are chunky (rather than needles), don't stick to the bottom, consistently diffract to high resolution, tolerate high solvent concentrations, don't grow skin on the drop, and don't require complicated cryoprotection. Nevertheless, non-ideal crystals are feasible too: it simply makes soaking, harvesting and data collection slower (further streamlining is in progress, prioritised according to users needs).
The best way to address prohibitively non-ideal crystals is by finding different crystal forms, whether by using different crystallization conditions, construct redesign, surface mutations, or whatever other trick works.
Full details of our fragment libraries are available here (or click on menu left).
Diamond Light Source is the UK's national synchrotron science facility, located at the Harwell Science and Innovation Campus in Oxfordshire.
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