Drug Discovery

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.

(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

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.

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.

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

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.