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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].

Figure 1: 1H-15N g-HNMQC and 19F-15N g-HNMQC experiments.

Figure 2: 1H-29Si g-HMQC experiment.

 

The selected compound(s) will be moved to candidate development with scale-up of the synthetic route, and characterization of the resulting material.

NMR will play an important role in reaction monitoring to ensure, with other techniques, a full understanding of the different steps of the chemical steps with identification of by-products and impurities.

Hyphenated HPLC- NMR has been used in the example in Figure 3 for the identification of co‑eluting low‑level impurities in key intermediate; Spectrum A has been acquired after injection of the mother liquors while Spectrum B has been acquired after injection of 100 µL of a solution of key-intermediate. Detailed analysis on the impurity in the mother liquors with a time-slice HPLC-NMR experiment (3 spectra at 10 sec. interval during peak elution) allowed the confirmation that the impurity was in fact a mixture of two co-eluting products. Structures determination has then been obtained after purification using standard NMR experiments [2].

 

Figure 3: Identification of co-eluting low-level impurities.

 

Critical experiments are also required in the case of UV transparent compounds, which will not be monitored by classical chromatographic techniques as reported in Figure 4 [2].

 

Figure 4: Reaction monitoring: Continuous-flow HPLC-NMR.

 

The final API will be fully characterized to profile the solid state profile, and to support the formulation studies. In addition to the solution phase NMR, solid-state NMR (ssNMR) will be used together with a variety of techniques to ensure a full understanding of compound behaviour.

An interesting application of solution NMR is reported in Figure 5 where experiments have been progressed for the determination of the critical micelle concentration (CMC) (value of the solute concentration at which half the total solute is present in the free monomeric form). NMR spectroscopy can be an alternative method to measure the CMC value, being the chemical shift concentration-dependent, particularly in the case of solute-solute intermolecular interactions, with typical downfield shifts of ¹H NMR resonances on dilution.

 

Figure 5: Critical Micelle Concentration (CMC) Determination.

In the example, the particularly large shielding for the aromatic protons allowed the assumption that the aromatic rings of the studied molecules that constitute the aggregate are placed in the inner hydrophobic part of the micelle, while the N-acetylpiperazine ring is somehow representing the hydrophilic external surface of the micelle itself. The forces that are involved in the aggregation are then those typical of π-staking. The CMC can then be evaluated plotting the chemical shift variation (Δδ, ppm) versus the reciprocal of the concentration (L/mol). No significant chemical shift variation was observed in the solutions at concentration ≤ 1 mg/mL, while a linear trend was observed in the concentration range 50 ÷ 3 mg/mL. Thus, assumption could be made that the intercepts of these lines on the x axis corresponded to the 1/CMC value. NMR measurements performed at 15 °C, 25 °C, 35 °C and 45 °C allowed the temperature dependence of the CMC to be determined and the thermodynamic parameters of the micellization process to be extrapolated [4].

In Parts 1 and 2, a few examples of the possibilities of the NMR techniques to support the drug discovery and development have been made with a focus on structures determination and characterization. The impact of NMR techniques on in vitro , ex vivo , in vivo and clinical phases will be covered in Part 3.

References 

1. C. Marchioro, Spectroscopy Solutions , 3 (1), (2015).

2. S. Provera, C. Marchioro, unpublished data.

3. S. Provera, S. Davalli; G. H. Raza; S. Contini; C. Marchioro, Magn. Reson. Chem. , 39, 38 (2001).

4. S. Provera, S. Beato, Z. Cimarosti, L. Turco, A. Casazza, G. Caivano, C. Marchioro, J. Pharm. Biomed. Anal. , 54, 48 (2011).

Carla Marchioro

Scientific Director at R4R & Head of Discovery and Development; Chief Technology & Operations Advisor at AnCoreX

Verona Area, Italy

Pharmaceuticals

Current	AnCoreX Therapeutics, R4R
Previous	Aptuit, GlaxoSmithKline
Education	Università degli Studi di Padova

https://www.linkedin.com/in/carlamarchioro

Carla Marchioro – ResearchGate

www.researchgate.net/profile/Carla_Marchioro

Carla Marchioro is Scientific Director, and Head of the Pharma & Analytical Division at Research for Rent, R4R, Italy where she is now after covering related positions in Aptuit and GlaxoSmithKline R&D where she has been leading multidisciplinary and cross national groups. In addition, she is also Chief Technology & Operations Adviser at AnCoreX Therapeutics.

She is an NMR expert with a chemistry background and has large experience in structural techniques. Over the years, she has developed an extended experience in a large part of the Research & Development process from target identification and progression to NDA filling.

In her group, in addition to classical structural and analytical approaches, state of the art techniques and technologies such as “omics”, computer-assisted drug design, fragments base screening, analytical and preparative SFC, quantitation by NMR, ssNMR methods for cells & tissues and more have been introduced and developed.

In addition to the R4R role, she is a member of a number of Scientific Boards, European and National Research funding bodies; and she has been part of the Scientific Advisory Board of the ProtEra company up to February 2010.She is author of a number of publications and presentations and she is a well-recognized member in the scientific community. She has been a member of the ENC Scientific Board, the chair of the 51” ENC (2010), member of the SMASH Conference Board, and the chair of the SMASH 2013 Conference.

Structural & Analytical expertise in the Drug Discovery, Chemical Development and Pharmaceutical Development Departments (up to transfer to Manufacturing groups). In addition, experience in the drug design and understanding of mechanism of action, metabolic pathways and safety related aspects.

Specialties: full understanding of mechanism of actions; full understanding of chemical and biological pathways; software and hardware design and needs; international experiences crossing countries and cultures.

Experience

Chief Technology & Operations Advisor

AnCoreX Therapeutics

May 2014 – Present (1 year 2 months)

Scientific Director & Head of Discovery and Development

R4R

January 2013 – Present (2 years 6 months)

Scientific Liaison Director

Aptuit

September 2011 – December 2012 (1 year 4 months)Verona Area, Italy

Director, Head of Structural & Analytical Scientific Strategy

Aptuit

October 2010 – December 2012 (2 years 3 months)

Director Analytical Chemistry

Aptuit

July 2010 – October 2010 (4 months)

Director & Site Head of Verona Analytical Chemistry

GlaxoSmithKline

January 2009 – June 2010 (1 year 6 months)

Objective of the Verona group was to provide Structural & Analytical expertises to the Verona/Harlow Centre for Excellence in Drug Discovery (Neurosciences CEDD), Chemical Development and Pharmaceutical Development Departments (up to transfer to Manufacturing groups) at the Verona GSK site. In addition, to contribute to the international initiatives of Molecular Drug Discovery (MDR) and Analytical Chemistry.

Director & Site Head of Verona & Zagreb Analytical Chemistry

GSK

January 2007 – December 2008 (2 years)

Objective of the Verona group was to provide Structural & Analytical expertises to the Verona/Harlow/Zagreb Centre for Excellence in Drug Discovery (Neurosciences CEDD), Chemical Development and Pharmaceutical Development Departments (up to transfer to Manufacturing groups) at the Verona GSK site. In addition, to contribute to the international initiatives of Molecular Drug Discovery (MDR) and Analytical Chemistry.
Objective of the Zagreb group was to provide Structural & Analytical expertises to the Zagreb Centre for Excellence in Drug Discovery (MacrolidesCEDD), and Pharmaceutical Development Departments at the Zagreb GSK site. In addition, to contribute to the international initiatives of Molecular Drug Discovery (MDR) and Analytical Chemistry.

Computational, Analytical and Structural Chemistry Verona Director

GlaxoSmithKline Verona

2000 – 2006 (6 years)

Director

GlaxoWellcome

1999 – 2000 (1 year)

Honors & Awards

Additional Honors & Awards

Contract Professor, Ferrara University (1999–2003);
Chiar for the SMASH2003 Conference, Verona, Italy
Chair for the 51th Experimental NMR Conference (ENC) (2010, Daytona Beach, US)
Chair for the SMASH2013 Conference, Santiago de Compostela, Spain

Publications

Discovery Process and Pharmacological Characterization of a Novel Dual Orexin 1 and Orexin 2 Receptor Antagonist Useful for Treatment of Sleep Disorders(Link)

Bioorganic & Medicinal Chemistry Letters (2011), 21, 5562

September 1, 2011

A novel, drug-like bis-amido piperidine derivative was identified as a potent dual OX1 and OX2 receptor antagonists, highly effective in a pre-clinical model of sleep.

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