Fragment-based drug design (FBDD) has led to a revolution in contemporary drug discovery, facilitating a bottom-up approach to the identification and evolution of bioactive small molecules into viable drug candidates. FBDD has thus far resulted in seven FDA-approved drugs and more than 50 clinical trials.
NMR spectroscopy is considered to be the gold standard method for fragment screening. In this context, fragment screening can be understood as the identification of a protein’s interaction with small molecules of molecular mass below 300 g/mol.
NMR fragment screening had previously necessitated the use of a high-field NMR spectrometer, but recent developments by NexMR have enabled the screening of fragment libraries on a cryogen-free benchtop NMR spectrometer; for example, Oxford Instruments’ X-Pulse.
NexMR’s technology illuminates samples to better enhance the NMR signal of photoinducible fragments, allowing these to be detected within seconds to minutes.
This improved sensitivity is realized by spiking the sample with a photosensitizer and an oxygen-quenching enzyme cocktail. The sample is illuminated for several seconds while sitting in the magnet.
Photo-induced signal enhancement is the most rapid means of improving NMR instruments’ sensitivity. It can also be performed at room temperature in an aqueous buffer - a highly preferable condition for drug discovery.
Fragment screening requires that sample illumination be compatible with an automated sample changer. This ensures high enough throughput to take full advantage of the ultrafast measurement capabilities of NexMR’s photoinducible fragment libraries.
Standard benchtop NMR setups are unsuited to illumination via automated sample changers. Manual intervention is required because the light coupling requires a physical connection between the light source and the sample via a waveguide.
A customized version of the X-Pulse is available, which features integrated light capability alongside an automated setup process. This setup can accommodate a screening rate of one sample every five minutes on a benchtop NMR spectrometer, the equivalent of 288 samples per day.
Oxford Instruments’ X-Pulse is a modular broadband benchtop spectrometer that offers unparalleled flexibility to virtually any analytical or research laboratory. As well as its capacity to accommodate a high-throughput autosampler, the X-Pulse can also be equipped with a flow cell or variable temperature (0 °C to 65 °C).
Figure 1. Automated X-pulse with light and measurement of the signal-to-noise. Image Credit: Oxford Instruments NMR
Performance of NexMR’s fragment kits with X-Pulse
NexMR’s fragment libraries (NMhare) contain a range of aromatic scaffolds, all of which can be observed using an X-Pulse benchtop NMR spectrometer with integrated light capability.
Following a few seconds of illumination, low concentrations of the fragments in an aqueous buffer (200 µmol/ℓ) can be detected within a single two-second scan (Figure 2).
In the example presented here, a total of 16 of the 18 tested fragments demonstrated a notable signal-to-noise ratio (SNR) following a single scan at 200 µmol/ℓ.
The median SNR was determined to be seven (with an average of 20), while the maximum observed SNR with a single scan was 112, and the minimum was two.
It was noted that an accumulation of 16 scans results in a minimum observable SNR of 10, allowing for quantitative peak analysis. The maximum SNR was determined to be 450; with a median of 30, and an average of 82.
Without photo-boosted NMR, these types of experiments would take more than 100 hours, but this level of performance enables fragment screening within just two minutes.
Figure 2. Photo-boosted NMR spectra (blue) of three fragments with diverse aromatic scaffolds after 2 seconds of illumination and the same experiment without illumination (black). Image Credit: Oxford Instruments NMR
Figure 3. Signal-to-noise ratio (SNR) per molecule and scan. Each molecule was measured at a concentration of 200 μmol/ℓ. Image Credit: Oxford Instruments NMR
Low micromolar measurements
For quantitative peak analysis, an SNR of 10 is typically required. Ligand-observed NMR screening is performed at 50-200 μmol/ℓ ligand concentrations at high magnetic fields.
In the example presented here, SNR was evaluated at different concentrations for tryptophan and cresol - two emblematic molecules yielding photo-boosted NMR.
Sixteen scans were performed in two minutes, confirming that it was possible to observe down to 20 μmol/ℓ for each molecule (Figure 4). For quantitative analysis, it is advisable to use 64 (eight-minute) scans for 20 μmol/ℓ screening and 16 scans for 50 μmol/ℓ screening.
Figure 4. Measurement of photoinducible reference fragments at different micromolar concentrations. Image Credit: Oxford Instruments NMR
Fragment screening with the X-Pulse benchtop NMR spectrometer and NMhare photo-inducible libraries
Small molecules have the potential to interact with larger macromolecules, forming a complex in a slow rotational motion. Under these circumstances, small molecule polarization is transferred to the protein via the nuclear Overhauser effect (NOE). This results in a reduction in signal intensity.
Identifying a protein-fragment binding event is possible by tracking the signal reduction after a protein of interest has been added. This fragment screening technology has been documented previously by NexMR and the Riek Lab (ETH Zürich).1 The signal reduction can be quantified with the polarization ratio (PR). This is defined as:
In the formula shown here, IPL and IL refer to the integral of the hyperpolarized ligand peak with and without protein, respectively.
The molecules here were tested against known protein targets to observe the signal reduction. This was done by binding all molecules that exhibited photo-boosted signals.
One fragment was considered a ‘hit,’ because this showed an especially significant reduction in the signal. It was noted that the hit was seen for the PIN1 protein, but the KRAS protein exhibited a characteristic decrease in signal.
Should PIN1 have been a druggable target protein, medicinal chemists would likely begin to base their drug design around this fragment because it had been shown to bind with the target protein.
Figure 5. Fragment screening of two fragment molecules against KRAS and PIN1, two oncoproteins. The polarization ratios (PR) are shown to reflect quantitative analysis. Image Credit: Oxford Instruments NMR
Summary
This article highlighted the potential of NexMR’s fragment library and Oxford Instruments’ X-Pulse with autosampler to enable automated high-throughput fragment screening.
Image Credit: Oxford Instruments NMR
Using photo-induced signal boosting, the experiment time was reduced from over 100 hours to two minutes. This significant reduction enabled the testing of fragments for their photo response and subsequent screening of protein molecules for binding, all within a single working day.
This highly reliable, high-throughput screening approach can enable academic and industrial pharmaceutical researchers to accelerate drug development in their laboratories while leveraging the many benefits of small-footprint, cryogen-free benchtop NMR.
References and further reading
- F. Torres et al., J. Am. Chem. Soc., 2023, 145, 12066-12080.
Acknowledgments
Produced from materials originally authored by Oxford Instruments.
About NexMR
NexMR is a technology company developing and commercializing fragment libraries tuned for photohyperpolarization and an experimental protocol for fragment screening and affinity determination. NexMR’s technology enables ultrafast detection of micromolar amounts of small molecules, even at low magnetic fields. For more detailed information: https://www.nexmr.com Contact: [email protected]
About Oxford Instruments NMR
Oxford Instruments offers a range of Nuclear Magnetic Resonance (NMR) instruments to multiple industries. Our portfolio includes, X-Pulse, a high-resolution cryogen-free broadband benchtop NMR spectrometer. Combining true broadband X-nuclei capability, flow chemistry, reaction monitoring and variable temperature with superior spectral resolution, X-Pulse lets you perform a wide range of experiments on the bench in your lab.
The MQC+ range of benchtop NMR analysers offers broad applications across the agriculture, foods, consumer products, textiles, and polymer industries. MQC+ analysers are used to measure oil, water, fluorine, and solid fat in a variety of samples and are typically used for quality assurance and quality control. To increase laboratory productivity and efficiency, MQC+ analysers can be used with the MQ-Auto sample automation system. MQR is a low-resolution, high-performance TD-NMR research system designed for applications based on relaxation and/or diffusion measurements. Our GeoSpec NMR core analysers are designed specifically for studies of core samples from oilfield reservoirs, with installations in almost every major oil producer and SCAL laboratory world-wide.
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