Fragment-based screening is now well-established as a powerful approach to early drug ("lead") discovery.
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XChem: X-ray structure-accelerated, synthesis-aligned fragment medicinal chemistry
As part of Diamond’s effort to combat COVID-19 (as demonstrated by the fragment screening campaign against SAR-CoV-2 Main protease and Nsp3 macrodomain), fragment screening expertise and infrastructure are offered to users working on Covid-19 related projects.
Due to access/travel restrictions, experienced XChem staff will perform the preparation of the samples on-site and collect the data remotely unattended.
Project pre-requisites (XChem staff are available to discuss these criteria and how to best achieve them):
Robust crystallisation condition that yields reproducible high quality crystals in Swiss-Ci 3-drop plates (reservoir volume of 30 ul and drop size of 200-600 nl);
Ideally (but not restrictive), of the 288 crystallisation drops in the plate, more than 50% will have crystals of reasonable diffraction quality (2.6 Å) and at least 35 um size;
Consistent diffraction quality of crystals (ideally all crystals tested diffract to 2.6 Å or better);
Structural accessibility of the site of interest, i.e. the site of interest is not involved in the crystal packing or blocked.
Available libraries can be found here
The most commonly used libraries are the DSI-poised, FragLite, Mini Frag and York3D libraries
Fragment screening experiments:
All fragments will be individually soaked in the crystal system using an optimised condition that will be defined in preliminary experiments.
What you will need to provide:
If you are located in close proximity to Diamond Light Source then delivery of swissCI-3drops crystallisation plates with crystals delivered is preferred.
Contributing physical compounds related to COVID-19 targets for XChem screen:
for Mpro, we have a defined workflow as part of the Moonshot project (see details here).
for other targets, please contact the owner of the protein target first and Frank and Alice. then please send the compounds as DMSO solutions. We recommend 4 uL at 100mM or higher in 1536ldv (LP-0400/LP-0400-BC) labcyte plate (more information available here). If you don’t have echo-compatible plates, any other plate type or format is possible, as long as the sample is completely diluted and shipped on dry-ice. Most imporantly, please send us the corresponding plate map with compound ID, SMILES string, concentration and volume following this format.
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 1000 compounds to be screened individually in less than a week (including 36 hours' unattended beamtime). The process covers soaking, harvesting, automatic data collection, and data analysis; fragment libraries are available, though 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 user community is growing and you can follow current discussions in the XChem bulletin board.
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 access is governed by the same usage policies as the rest of Diamond's user programme. In particular, users are required to publish their results and deposit their hit structures in the PDB, as for all MX beamtime. In addition, we may occasionally request brief reports on how hits are progressing to potent compounds, to help us evaluate the effectiveness of the facility.
Academic access is not limited to academic groups; industry-based users can also apply by the same peer-review route, but will then be expected to publish and deposit just like academic groups. (This is in contrast to commercial access via the Industrial Liason Office, which is incidentally also available to academic groups provided they are prepared to pay commercial rates.)
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 (though still under evaluation).
We provide fragment libraries (or click on menu), 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.
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