February 4, 2013
In a recent LinkedIn Discussion post, Ben Davis posed the following question:
"Do any of the commercially available fragment libraries come with reference 1D NMR spectra acquired in aqueous solution?"
Most commercial vendors of fragments do not offer nuclear magnetic resonance (NMR) reference spectra with their compounds useful to fragment screeners; if anything, it is 100% organic solvent, at room temperature, at relatively low magnetic field strength. The NMR spectra of fragments and other small molecules are very solvent- and sample-dependent; different buffers, solvents, temperatures and magnet field strengths can generate large spectral differences for the exact same compound. As a result, NMR reference spectra acquired for fragments in organic solvent cannot be used to design fragment mixtures, one of the key advantages in NMR screening. Furthermore, solubility in organic solvent is no measure of solubility in the mostly aqueous buffer conditions typically used in NMR-based fragment screening.
At Emerald Bio, the leading protein resource, we routinely acquire NMR reference spectra for all our commercially-sourced fragment screening compounds as part of our quality control (QC) procedures. This is necessary to ensure the identity, the purity and the solubility of each fragment we use for screening campaigns. This data are further used to design cocktails of 9-10 fragments with minimal peak overlap for efficient STD-NMR screening in-house.
Recently, we selected a random set of commercial fragment compounds for analysis, and closely examined those that failed to better understand the reasons behind it. The most common reason for QC failure was insolubility (47%), followed by degradation or impurities (39%), and then spectral mismatch (17%) [Note: Compounds can acquire multiple QC designations, hence total incidences > 100% ]. Less than 4% of all compounds assayed failed due to solvent peak overlap or lack of non-exchangeable protons, both requirements for NMR screening. Failure rates were as high as 33% per individual vendor, with an overall average of 16% (see Figure 1). Although some vendor fragments yielded failures, ordering very few compounds from a single vendor did not change the outcome (8% from over 20 vendors ordering 10 or fewer fragments from each).
These results highlight the importance of implementing tight quality control measures for preliminary vetting of commercially-sourced materials, as well as maintaining and curating a fragment screening library. It also puts forth a statistical likelihood of around 10-15% failure, regardless of vendor. Most importantly, we have seen our methods reduce risks while accelerating drug discovery. Do these numbers make sense to you? How do they measure up with your fragment library?
Let us know what you think.
Figure 1. Percentage of NMR QC failure rates for a random set of commercially available fragment compounds. Fragments were first dissolved to 50 mM in deuterated dimethyl sulfoxide (d6-DMSO). Final NMR samples were 1.0 mM fragment in 50:100:350 µL d6-DMSO:D2O:low salt aqueous buffer. NMR data were acquired at 10°C at 500 MHz field strength.
April 11, 2012
The relatively new field of Fragment Based Drug Discovery received a much-needed boost late last year when the FDA approved the first drug whose genesis was screening of a fragment library (see reference below). The BRAF inhibitor Zelboraf (vemurafenib) grew out of biochemical screening of a panel of kinases at Plexxikon, followed by intensive X-ray crystallographic analysis of hits from the primary screen.
There are, however, many different methods that FBDD practitioners can employ in screening. If an amenable crystal system is available, soaking of crystals and XRD analysis can be performed. Ligand-observe NMR methods such as saturation transfer difference (STD-NMR) are also often used. SPR, thermal shift, and a variety of biochemical methods are likewise well-documented.
What methods should you use? The answer is usually complex, and depends on the nature and abundance of your target macromolecule, as well as the size of the fragment library you wish to screen. At Emerald, we currently rely heavily on X-ray, STD-NMR, SPR and thermal shift techniques, and we prefer to use more than one technique at a time to aid with prioritization and characterization of hits. There are a couple of upcoming opportunities to have this discussion in real time…
We are looking forward to meeting lots of our past and future collaborators at the Drug Discovery Chemistry (DDC) conference in San Diego April 16-19. Alex Burgin, CSO of Emerald BioStructures, will be giving a workshop as part of the FBDD session on April 16. He will be co-teaching a short course with Daniel Erlanson (author of the excellent blog practical fragments) entitled “Advanced Tools and Technologies for Fragment-Based Design." If you would like to speak personally with Alex, he will be available for the remainder of the week in San Diego. Additionally, Emerald is holding an informal MeetUp at the conference on April 17, from 6;30-8:30 PM. Interested parties should contact Diana Wetmore for location details.
New fragment screening methods are constantly evolving. One technology we find particularly intriguing is the “CEfrag” Capillary Electrophoresis technique of Selcia, Inc. This approach monitors the mobility shift of a probe ligand as both target and a cocktail of fragments move through a gel-filled capillary. The image below is a schematic of the technology from the Selcia Discovery website.
In addition to presentations at DDC, a scientist from Selcia will give a full description of their fragment screening method as a guest presenter in the Emerald BioStructures Webinar series. The webinar, titled "Complementing Biophysical and Structural Methods for Drug Discovery with Capillary Electrophoresis," can be watched live on April 17 at 11 am Pacific Time, or as an archived presentation any time after that. If you watch live there’ll be opportunities to send in questions for discussion at the end of the webinar.
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