Carla Marchioro

Introduction

As is well known the drug discovery and development process is a complex process typically starting from the target identification and validation of a target to progress to clinical studies and hopefully ending with a new drug to the market [Figure 1].

 

 

In this process, different approaches and methods are required to understand the disease’s mechanisms, to profile hit molecules that will be progressed to leads suitable for full scale lead optimization programmes and then to generate quality drug candidates to advance to clinical studies.

Focusing on small molecules’ drugs and on hit to candidate phases, a variety of techniques will be used to study the compounds’ profiles at different levels including the physicochemical profiles, purity, solid state behaviours and structures to ensure a quality hit/lead/candidate and related data to allow understanding of the mechanism of action and SAR correlation.

Nuclear Magnetic Resonance (NMR) spectroscopy will play a pivotal role generating data on the molecular interactions between ligands and biological targets, in addition to providing the structures of drug molecules, by-products, impurities, metabolites and quantification data.

NMR Screening Impact 

During the drug discovery phase, NMR spectroscopy is becoming more and more relevant with application at multiple stages along the progression of a project: NMR experiments are used for hits generation, lead discovery and optimization, evaluation of in vitro/in vivo selectivity and efficacy, studies drug toxicity profiles and identification of new drug discovery targets.

Over the last years there has been a large increase in the application of NMR techniques for the rapid determination of protein-ligand structures and interactions, to powerfully screen fragment-based libraries, to identify biological relevant ligand interactions, and to monitor changes in the metabolome from bio-fluids and cells to explore compounds activity.

Focusing on the NMR-based screening techniques, the NMR experiments could be divided into two main categories: target observed and ligand observed methods.

Without doubt, high resolution protein structure is a key requirement to evaluate the biological relevance of a hit from screening and HTS (high-throughput screening) and NMR together with X-ray are playing an essential role.

The last period has witnessed the generation of fast NMR sequences and methods to allow a faster impact on the drug discovery project time but the NMR target-observed techniques still require time, material, possible labelling and difficulties in handling a number of different hits and studies on mixtures.

Nevertheless, if the resonance assignment of the labelled target is known, the exploitation of differences in chemical shifts between free and bound target in two dimensional correlation spectra (shift mapping) will provide important structural information on the site of binding. The experiments could be also be of high value on selectively labelled target decreasing the spectra complexity and so increasing the size of the target that could be studied by NMR techniques.

Considering now that the chemical shift is highly sensitive to the environment of the atom and, as a consequence, it provides information on the binding of a small molecule to a biological target, and on which part of the molecule is interacting and where, it is clear that ligand-observed techniques could generate proof and data for the binding understanding and profile.

In addition, other experiments based on molecule relaxation values are sensitive to the motion of the compound (free vs bound state) and together these experiments will allow validation of ligand binding and/or identification of ligands also in mixtures.

The ligand observed techniques benefit of:

• one-dimensional experiments;
• detection of the ligand’s signals (facilitating also the mixture analysis);
• smaller amount of target substrate;
• structural and binding information of the ligand;
• detection of week binding ligands;
• limited restrictions on size and type of the target, with no isotope labelling requirement or target information details.

On the undesirable side, the techniques could generate false negatives (strong binding and slow exchange equilibria) or false positives (unspecific binding) but all these aspects could be further studied to result in a substantiated answer.

During the Hits generation phase, NMR will be used for the determination of binding affinity values toward the hit validation step to generate a lead where the NMR experiments will also remove the false positives and locate the binding site (for example within the FBDD approach). In the lead optimization phase, to improve potency of the compounds, the epitope mapping will be determined, together with the conformation of the bound ligand, while in the late lead optimization stage for the candidate selection the NMR will support the bioavailability, ADME, PK and toxicological experiments.

The combination of NMR screening methods with other techniques, such as in silico computational protocol, X-ray crystallography, and biophysical experiments will decrease the number of compounds to be studied generating filters and resulting in time and cost saving and efficiency increase. The NMR will be so used to screen and profile a library (or set) of compounds with the unique ability of providing proof for binding between the ligand and the biological target and subsequently being able to detect the binding site and determining the construct of the complex.

This short note will not include technical details of the many NMR-based methods that could be found in several papers and reviews across the last decade [1-8].

The versatility of the NMR techniques is allowing the detection of target-ligand interactions through a large variety of measurements. The insights will derive from the observation of peak intensity and/or line-width changes, saturation transfer differences (STD), chemical shifts perturbations, R2 relaxation effects, R1, sel competition data, induced transferred NOEs, interligand NOEs, diffusion coefficient measurements and changes, and, in general, from monitoring any changes in the NMR spectra resulting from the ligand-target interactions.

A big impact of the NMR techniques is also evident on the “undruggable” targets when other techniques alone fail to result in relevant data and studies on protein-protein, protein-membrane macromolecular recognition are now becoming more and more frequently successfully progressed [9].

The lead compound will need then to be optimized in the bioavailability, efficacy and toxicity profile to result in a candidate to be progressed to in vivo studies, in animals, and finally on humans.

NMR will contribute heavily in all these phases will full characterization of the compound and solid state data, stability studies, formulation studies and NMR-based metabolomics experiments. All these aspects will be covered in a future contribution.

References 

1. M. Pellecchia, I. Bertini, D. Cowburn, C. Dalvit, E. Giralt, W. Jahnke, T.L. James, S.W. Homans, H. Kessler and C. Luchinat, Nat. Rev. Drug Discovery, 7, 738 (2008).

2. R. Powers, Expert Opin. Drug Discov., 4(10), 1077 (2009).

3. R. Powers, J. Med. Chem., 57(14), 5860 (2014).

4. M.J. Harner, A.O. Frank and S.W.Fesik, J. Biomol. NMR, 56(2), 65 (2013).

5. C. Dalvit, Prog. Nucl. Magn. Reson. Spectrosc., 51, 243 (2007).

6. M. Mayer and B. Meyer, Angewandte Chemie Int. Edition, 38,1784 (1999).

7. P.J. Hajduk, D.J. Burns, Comb. Chem. High Throughput Screen., 5, 613 (2002).

8. W.Jahnke and D.A. Erlanson (Editors), Fragment-based Approaches in Drug Discovery, Wiley-VCH, 2006.

9. D.M. Dias, I. Van Molle, M.G.J. Baud, C. Galdeano, C.F. G. C. Geraldes and Alessio Ciulli, ACS Med. Chem. Lett., 5 (1), 23 (2014).

In Part 2 of this series, Carla Marchioro continues to offer her insights into the contribution of NMR structural techniques to the drug discovery and development process.

 

Introduction 

After some insights on the impact of NMR techniques on the initial drug discovery phase [1], NMR techniques applied in the progression of a compound from lead to candidate and to drug are clearly having, together with other techniques, a large impact with full structural determination, full understanding of chemical reactions, studies of molecules’ behaviour in solutions and solid states and stability monitoring with determination of by-products.

NMR Techniques in Lead Optimization and Drug Development 

As soon as a compound has been identified as a lead to be progressed to the candidate phase, several NMR studies will be required to support the chemical effort, and to ensure a quality profile of the selected compound.

Synthesis of different compounds will be progressed to obtain the desired biological profile and structures will be characterized and studied to also support the computational effort, and to monitor and determine the purity for the biological tests.

Several techniques will be used, such a MS, IR, HPLC,…, to results together with the NMR data in a full profile of the studied compound.

Classical mono- and two-dimensional NMR techniques (¹H and ¹³C) will be performed and, if required, experiments on additional nuclei will add further information to the full structural determination. As an example, in Figures 1 and 2, ¹H-¹⁵N g-HNMQC, ¹⁹F-¹⁵N g-HNMQC, and ¹H-²⁹Si g-HMQC have been used to obtain the full structures characterizations [2, 3].