Drug Discovery

The research that goes into Drug Discovery, or the search for new potential medications, is an extensive process through which new therapeutic entities are identified and extensively studied. How therapeutics produce their targeted effects, is examined from the tissue level to the cellular level and all the way to the structural level, where structural information provide invaluable insights on the atomic interactions between a drug and its target. This information is exploited in optimizing a drug’s potency while reducing its off-target effects. JEOL Cryo-Electron Microscopes and Nuclear Magnetic Resonance Spectrometers are instrumental in structure-based drug design, while JEOL Scanning Electron Microscopes aid with quality control aspects of this field.

Cryo-Electron Microscopy 

The JEOL CRYO ARM is accelerating drug discovery and development of novel therapeutics with its stability, ultrahigh throughput and resolution, and unattended operation. The CRYO ARM routinely produces near-atomic resolution 3-dimensional structures of proteins, biological macromolecules and assemblies allowing researchers to develop targeted therapeutics. The increasing resolution achieved with the CRYO ARM has been reported in an extensive bibliography. In 2023 a 1.19A structure of apoferritin, a protein found in the intestinal mucosa membrane, was reported by Maki-Yonekura et al, demonstrating that the JEOL CRYO ARM can produce structures at true atomic resolution.

MicroED

Until the resolution revolution of Transmission Electron Microscopy, the ability to solve high resolution biological structures was only possible using a diffraction technique, X-ray Crystallography. Now a novel technique, MicroED, borrows from X-ray crystallography but uses electrons as the incident beam, allowing atomic details to be acquired from much smaller crystals, as small as 200nm or less in size. The JEOL CRYO ARM is fully equipped with a diffraction beam setting, a beam stopper, and an ultra-stable rotating stage for microED data collection.
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Nuclear Magnetic Resonance

Structure analysis of active pharmaceutical ingredients with XtaLAB Synergy-ED and JEOL MS and NMR

Structure analysis of active pharmaceutical ingredients with XtaLAB Synergy-ED and JEOL MS and NMR
XtaLAB Synergy-ED is a fully optimized electron diffractometer for microcrystals analysis. The key feature of this product allows molecular structural analysis of pharmaceutical raw materials such as active pharmaceutical ingredients with powder. In addition, JEOL mass spectrometer (MS) and nuclear magnetic resonance (NMR) spectrometer provide detailed information for determining the molecular structure.

Electron diffraction structure analysis of active pharmaceutical ingredient powders

Electron diffraction structure analysis of active pharmaceutical ingredient powders
XtaLAB Synergy-ED allows electron diffraction structure analysis of powder samples less than few μm without sample preparation. CrysAlisPro for ED is an instrument control and single crystal analysis software platform for 3D ED/microED experiments. This system provides researchers a simple and effective platform for electron crystallography. The example above shows the results of electron diffraction structure analysis of active pharmaceutical ingredient powders. The measurement time required to complete the data collection is few minutes. It is possible to use Cryo-transfer holders for heat sensitive samples and hydrates.

Structure analysis of Voriconazole powder

Structure analysis of Voriconazole powder

Structure refinement is a method of obtaining accurate atomic coordinates by using electron diffraction intensities. In the example above, this refinement is performed by the full matrix least-squares method of SHELXL. In this method, a local minimum model with incorrect, but not necessarily unrealistic geometries can be achieved during refinement (shown left). In addition, NMR analysis provides the details of Voriconazole chemical structure (shown center). An initial model of Voriconazole determined by electron diffraction structural analysis can be refined with the chemical model of NMR analysis result (shown right). The left figure is 13C with 1H and 19F decoupling and 13C with 1H decoupling NMR spectra of Voriconazole by using JNM-ECZL 500R. ROYALPROBE™ HFX enables a wide variety of advanced 1H and 19F NMR experiments with dual tune mode. It is effective to simplify spectral assignments with 13C with 1H and 19F decoupling and many unique correlation experiments for the analysis of organic compounds containing fluorine atoms, for example Voriconazole.

Structure analysis of Reserpine powder

Structure analysis of Reserpine powder
In order to elucidate the molecular structure of Reserpine, the molecular formula is determined by using MS. In the example, the molecular formula of reserpine is provided from the Mass spectrum by using JMS-S3000 SpiralTOF™-plus 2.0 (shown top left). In addition, NMR analysis provides details of Reserpine chemical structure (shown bottom left). The right-hand is the molecular structure of Reserpine which is refined by the chemical structure model of MS and NMR results.

The merit of the observation of protein – ligand 1 interaction by NMR is that one can observe interaction at equilibrium state between association and dissociation in solution. 1D NMR techniques are useful methods that can be used to reveal the target molecule among candidates of many small compounds. In this context, they are attracting attention as screening 2 method s of pharmaceutical products.

1The ligand is a molecule that specifically binds to a specific receptor (protein).
2Screening is select those that show drug efficacy / activity from a large number of candidate compounds.

Spin Diffusion and Saturation Transfer

Spin Diffusion and Saturation Transfer
Fig 1: Explanation of saturation transfer

STD (Saturation Transfer Difference)

In STD experiment, one obtains the difference spectrum between a spectrum with saturation and a spectrum without saturation. As a result, signals that show no intensity change are canceled out and only binding ligand signals remain. In other words, STD spectrum shows only ligand signals with binding activity to protein. A schematic explanation of STD is shown in Fig 2. Red-colored area indicates saturation where signals are not observed or are observed with reduced intensity.
Fig 2: Explanation of STD

STD experiment

STD experiment produces a difference spectrum. The difference spectrum can be obtained by alternately collecting the excited spectrum and the non-excited spectrum in the same experiment. Moreover, protein signals are reduced by T1ρfilter to make analysis easier. (Fig 3)
The pulse sequence of STD experiment
Fig 3: The pulse sequence of STD experiment
(Reference: J. Klein, R.Meinecke, M.Mayer, and B.Meyer, J. Am. Chem. Soc. 1999, 121, 5336-5337.)

STD spectrum

Fig 4. shows the STD spectrum (A) and 1H spectrum (B) of mixture solution of Human Serum Albumin (HSA), L-(+)-Arginine (Arg) and L-Tryptophan (Trp). In the STD spectrum, Tryptophan signals which have binding activity to Albumin can be observed, while Arginine signals which have no binding activity are not present.
(A) STD spectrum, and (B) 1H spectrum mixture solution of Albumin 0.1mM，Trp 2mM，Arg 2mM /D2O
Fig 4: (A) STD spectrum, and (B) 1H spectrum mixture solution of Albumin 0.1mM，Trp 2mM，Arg 2mM /D2O

(A) STD spectrum: 64 scans, selective excitation position 0.6ppm, non excitation position -10ppm, saturation time 8s, T1ρ filter 50ms
(B) 1H spectrum: 64 scans
console: JNM-ECZ400S+ROYALPROBE

DIRECTION (Difference of Inversion RECovery rates with and without Target IrradiatiON)

DIRECTION1 is a NMR technique to analyze the protein-ligand interaction by longitudinal relaxation time. In general, signal strength in the STD spectrum increases proportionately to the strength of interaction with protein. Thus, it’s possible to obtain hints about the part of ligand involved in binding. However, when ligand’s T1 is short, efficiency of saturation transfer decreases. Therefore, STD spectrum analysis needs to take into consideration the T1 of each ligand signal. DIRECTION is the experiment that estimates the efficiency of saturation transfer by probing not the signal intensity but directly the T1. DIRECTION is essentially an inversion recovery experiment where T1 is measured twice with saturation transfer and no saturation transfer. 1H signals from ligands that strongly bind to protein show decreased T1.

DIRECTION experiment

In DIRECTION experiment, T1 is measured twice with and without presaturation (Fig.5). Analysis of the changes in 1H’s T1 can identify binding compounds. Protein signals are decreased by T2 filter.

DIRECTION experiment
Fig 5: DIRECTION experiment
Reference: Y. Mizukoshi, A.Abe, T.Takizawa, H.Hanzawa, Y.Fukunishi, I.Shimada and H.Takahashi, Angew. Chem. Int. Ed. 2012, 51, 1362-1365.

Results of DIRECTION

Table 1 shows the results of DIRECTION for a mixture solution of 0.1mM HSA, 2mM L-(+)-Arg, and 2mM L-Trp / D2O. T1 of Arginine which has no binding activity shows no significant change when it is measured with and without saturation. On the other hand, Tryptophan's T1 is significantly decreased. Especially 3.22ppm and 3.24ppm's signals are drastically decreased.

DIRECTION: 32 scans, loop number 80, relaxation delay 15s, T 2filter 80ms, tau interval 0.1～16s 8 points console: JNM-ECZ400S+ROYALPROBE

assign CS/ppm no presaturation
T 1/s 		with presaturation
T 1/s 		Change %
1 arg 1.62 0.94 0.96 102.13
2 arg 1.77 0.96 0.94 97.92
3 arg 3.18 1.04 0.99 95.19
4 trp 3.22 1.11 0.62 55.86
5 trp 3.24 1.02 0.60 58.82
6 trp 3.38 1.06 0.70 66.04
7 arg 3.57 3.18 3.24 101.89
8 trp 3.93 1.83 1.28 69.95
9 trp 7.13 1.90 1.41 74.21
10 trp 7.21 1.92 1.41 73.44
11 trp 7.25 2.18 1.49 68.35
12 trp 7.48 1.92 1.22 63.54
13 trp 7.69 1.80 1.27 70.56
Table 1: Results of DIRECTION

For pharmaceutical formulation developments, probing binding form of a cocrystal between an active pharmaceutical ingredient (API) and a coformer is of great importance not only for realizing its properties but also for the intellectual property protection point -of-view. 1H solid state NMR using ultra fast MAS can be a powerful tool to probe hydrogen bond (HB) network and differentiate salts and cocrystals.

We report here an example of HB network analysis for a codrug formed by two APIs: namely theophylline (THEO) and pyridoxine·HCl (PyrH+Cl-). 1The HB characterization of the codrug is challenging, since five HB donors (three OH and two NH groups) and three acceptors (two C=O groups and one aromatic nitrogen) are present (Fig.1). In fact, single crystal X-ray diffraction could not access it due to the poor definition of the hydrogen atom positions. In order to probe the HB network and define binding form properly, we used two ver sions, J and D, of 14N-1H HMQC. 2-4The J-version is a simple HMQC experiment with rotor syncronous 14N observation in the indirect dimension through J-coupling and residual dipolar splittings (RDS), and mainly gives information of 14N-1H covalent bond. On the other hand, the D-version utilizes 14N-1H dipolar coupling reintroduced by a symmetry-based sequence (SR4), and observes longer-range N···H proximities. The combination of the two methods allows defining of the ionic character of the codrug, namely, salt or cocrystal.

The 14N-1H J- and D-HMQC spectra are shown in Fig.2a and 2b, respectively. The J-version spectrum shows only two correlation peaks, corresponding to covalently bonded N-H atoms (i.e., N1+-H1 and N7’-H7’). This suggests that there is no proton transfer between THEO and PyrH+Cl-, thus the codrug can be more properly defined as a salt co-crystal due to the ionic character of PyrH+Cl-. On the other hand, the D-version shows several correlations regarding with longer range 14N···1H proximities. Combination with 1H DQ MAS spectrum, which gives 1H-1H proximity information, elucidates the HB network described in Fig.3. This structure is supported and competed by X-ray data and DFT calculations.

Chemical structures of Theophylline (THEO) and Pridoxine(Pyr)H+Cl-
Fig.1 Chemical structures of Theophylline (THEO) and Pridoxine(Pyr)H+Cl-
Scheme of the main spatial proximities of THEO)/(Pyr)H+Cl- as found in the 14N-1H D-HMQC (blue), and the 1H-1H DQ MAS (red).

Fig.3 Scheme of the main spatial proximities of THEO)/(Pyr)H+Cl- as found in the 14N-1H D-HMQC (blue), and the 1H-1H DQ MAS (red).
(Reprinted with permission from ref 1. Copyright at 2018 American Chemical Society)

(a)14N-1H J-HMQC, (b)14N-1H D-HMQC, and (c)1H DQ MAS spectra for THEO/PyrH+Cl- obtained at 70kHz MAS.

Fig.2 (a)14N-1H J-HMQC, (b)14N-1H D-HMQC, and (c)1H DQ MAS spectra for THEO/PyrH+Cl- obtained at 70kHz MAS. (Reprinted with permission from ref 1. Copyright at 2018 American Chemical Society)

References

  • F. Rossi, P. C. Vioglio, S. Bordignon, V. Giorgio, C. Nervi, E. Priola, R. Gobetto, K. Yazawa, and M.R.Chierotti, Cryst. Growth Des., 2018, 18 (4), 2225–2233
  • S. Cavadini, A. Lupulescu, S. Antonijevic, and G. Bodenhausen, J. Am. Chem. Soc. 2006, 128, 7706
  • Z. Gan, J. Am. Chem. Soc. 2006, 128, 6040
  • Y.Nishiyama, Y. Endo, T. Nemoto, H. Utsumi, K. Yamauchi, K. Hioka, and T. Asakura, J. Magn. Reson. 2011, 208, 44-48
Learn more about JEOL NMR.

Scanning Electron Microscopy

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