Pharmaceuticals and Biotech

Pharma and biotech firms make wide use of synchrotron X-rays for drug discovery, drug development and drug delivery systems, as well as health care technologies such as implants and  radiotherapy. 

For drug discovery, the ESRF offers state-of-the-art protein crystallography facilities, including MASSIF, the world-leading unique beamline for the high throughput, fully automatic characterisation and data collection of macromolecular crystals. The ESRF produces more than half of the protein structures deposited in Europe. Thousands of samples are screened every day with high-throughput screening techniques.

Thanks to the automation of our facilities, protein crystallography measurements are possible through both mail-in and remote access from your home lab.

The other techniques most used by pharma and biotech companies are small/wide and ultra-small angle X-ray scattering, pair distribution function (PDF), micro- and nano-3D tomography and X-ray fluorescence and powder diffraction.

Characterisation of pharmaceutical active ingredients and formulas in situ and their behaviour in different conditions.

Studying drug delivery vectors such as nano-holders and phospholipid/micellular structures.

Resolving the crystal structures of drug formulation

CASE STUDIES

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Selective inhibition of the Nav1.7 pain channel; Crystal structure of a Nav1.7-antagonist complex viewed in a model membrane. Credits J. Payandeh.

Company

Genentech, Inc.

Challenge

Chronic pain is an important and unmet medical need that affects the quality of life for millions of people worldwide.  Available treatment options include opioids and non-steroidal anti-inflammatory drugs that can be effective but often have unwanted side effects, limiting their therapeutic utility.

Genetic studies in humans have recently identified a mutation in the voltage-gated sodium (Nav) channel, Nav1.7, which causes people to lose the ability to feel pain.  Intense efforts have been underway to identify antagonists that selectively inhibit only the Nav1.7 channel but leave the other eight human Nav channel isoforms unaffected.  It has been a major challenge to identify selective Nav1.7 channel inhibitors because all nine Nav channel isoforms are highly related in sequence, and all clinically available Nav channel blockers are poorly selective.

Sample

Nav1.7 contains four peripheral voltage-sensor domains (VSDs) that surround and control a central ion pore domain that allows sodium ions to enter and initiate action potentials in sensory neurons.  Researchers from Genentech, in collaboration with Xenon Pharmaceuticals, wanted to study a new class of inhibitors that appear to target the fourth voltage-sensor domain (VSD4) and selectively inhibit the Nav1.7 channel.  Because high-resolution structural studies of the human Nav1.7 channel are hindered by the molecules complexity, these researchers exploited a simpler bacterial Nav channel by fusing portions of human Nav1.7 (i.e. VSD4) onto it.

Solution

The Nav1.7 VSD4-bacterial channel fusion protein was crystallized and high-resolution diffraction studies were conducted at beamline ID29.  The resulting crystal structures revealed the details of how the new class of inhibitor directly interacts with residues within the fourth voltage-sensor domain to selectively and potently inhibit the Nav1.7 channel.

Benefits

The new structures provide insight into the mechanism of voltage sensing and can enable the design of more selective Nav channel antagonists.  Hopefully these results may accelerate the development of treatments for pain that selectively target Nav1.7 and aid drug design efforts aimed at other voltage-gated ion channels.

 

Science,  Vol. 350, Issue 6267, DOI: 10.1126/science.aac5464.

 

 

Companies

Technologie SERVIER and Novitom

Challenge

Curing is used to stabilise coatings to enhance tablet long-term stability and to ensure reproducible dissolution time. Technologie SERVIER wanted to investigate the effect of curing on the structure of a cellulose-based tablet coating, and in particular to determine the optimal duration for dynamic curing.

Sample

Tablets containing a freely-soluble drug were provided by SERVIER. The materials used to coat the tablets were commercially available. The coated tablets were dynamically cured for durations between 0 to 6 hours.

Solution

X-ray micro-computed tomography (XµCT) at beamline ID19 was used to examine the coating of the tablets. This non-destructive method revealed information about the internal porosity of the coating and provided observation of the qualitative differences between tablets with various degrees of curing. Regions of crystalline and amorphous matter were clearly visible along with the pore structure.

Reconstructed X┬ÁCT cross-sectional images obtained from uncured and cured coated tablets

Legend. Reconstructed XµCT cross-sectional images obtained from uncured and cured coated tablets and presented with drug dissolution profiles. The XµCT data were collected with a voxel size of 0.28 µm. Reprinted with permission from Elsevier.

Benefits

Using XµCT, the decrease in internal porosity was observed with increased curing time. The porosity volume provided a more precise determination of the optimal curing time than traditional dissolution testing alone.

Reference

Comprehensive study of dynamic curing effect on tablet coating structure, C. Gendre, M. Genty, J. César da Silva, A. Tfayli, M. Boiret, O. Lecoq, M. Baron, P. Chaminade, J.M. Péan, European Journal of Pharmaceutics and Biopharmaceutics 81, 657–665 (2012); doi: 10.1016/j.ejpb.2012.04.006.

Company

CRELUX GmbH (a WuXi AppTec Company)

Challenge

AMPK (AMP-activated protein kinase) is a Ser/Thr kinase composed of two regulatory subunits and a catalytic subunit  that together as a complex regulates the levels of energy in the cells. This complex is evolutionarily conserved and ubiquitously expressed. There are a total of 12 possible isoforms, which are distributed in the human body in a tissue-specific manner. For example, one isoform is highly expressed in skeletal muscles and a different  isoform is more specific to heart, brain or liver.  All in all, AMPK senses the energy levels of the cells (in the form of the so-called ATP) and allows upstream signals to activate it, in response to external nutritional stress.  AMPK substrates are involved in lipid metabolism, autophagy, mitochondrial biogenesis, and in the maintenance of glucose homeostasis. Therefore, this complex is a highly promising therapeutic drug target against diabetes, obesity, Wolff-Parkinson-White Syndrome, cancer, and aging.

There are, however, several challenges to generate soluble and stable complexes, and this is one of the many reasons why not many X-ray structures are available, especially at resolutions suitable to drive drug discovery efforts. Firstly, it is very complicated to be able to make crystals of the complex with the activated compound bound to it. Another obstacle is the extreme sensitivity of the tiny crystals to radiation damage. 

CRELUX/ WuXi AppTec, a company expert worldwide in premium drug discovery solutions for global pharma, biotech and research organizations, came to the ESRF to tackle this  challenge. 

Sample

CRELUX/WuXi AppTec used their expertise to design and produce a fully functional AMPK complex with the needed yield, purity, and specific post-translational modifications for successful crystallization and X-ray structure determination of an active complex. The AMPK sample contains an kinase activating compound and three AMP nucleotides (ATP-depleted scenario) bound to it.

Solution

CRELUX/WuXi AppTec scientists sent crystallized AMPK samples to the ESRF’s ID30-A beamline. Because of the complexity of the project, they needed a powerful beamline, state-of- the-art detector and a skillful scientist in-house to carry out the experiment. They managed to solve the structure of the complex at a resolution of 2.9 Angstroms, which was enough for CRELUX/WuXi AppTec to see the detailed chemical enviroment of the compound in the complex binding site. This corresponds to one of the highest resolution structure published so far for any AMPK isoform.

Benefits

The work at the ESRF will help CRELUX/WuXi AppTec to support their clients in the discovery and development of novel and more specific drugs that can influence AMPK activity in the cell and, as a result, adjust the energy balance in disease affected organs. Debora Konz Makino, Lab Head at CRELUX/WuXi AppTec, explains: ”Our long-standing collaboration with ESRF has greatly contributed to the success of most of our client's projects in the early stages of drug discovery. ESRF provides not only cutting edge infrastructure, but also excellent scientific support for X-ray data collection of biological macromolecules. We at CRELUX highly appreciate ESRF prompt and open communication.” 

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The AMPK structure solved. Credits: CRELUX.
 
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Envelope of the molecule calculated from the SAXS-data (top); conformation of the antibody as determined by X-ray crystallography (middle); Y-shaped conformation of a different antibody that fits to the SAXS-data (bottom).

Company

Boehringer

Challenge

Antibodies are a vital part of our immune system. These proteins bind to specific antigen proteins on the surface of foreign bodies such as bacteria and viruses in order to neutralize or disarm them. Since each antigen has a different shape, it requires a different antibody to attach to it. By tailoring antibodies to attach to proteins responsible for specific diseases, pharmaceutical companies seek to develop drugs that minimise side-effects caused by antibodies binding to the wrong targets. Researchers at Boehringer have been studying a molecule in an antibody and they found that it was unusually compact as a single crystal. The next step was to obtain structural information of the molecule in solution.

Sample

The antibody molecule in solution.

Solution

Using Small Angle X-ray scattering, the team managed to compare the measured scattering curve with that calculated from the X-ray structure they had previously solved. The results confirmed that the compact conformation does not exist in solution. Instead, the molecule adopts a Y-shaped conformation, commonly known for antibodies.

Benefits

SAXS experiments can help pharmaceutical companies to double-check the results they get using their own characterization techniques. SAXS is also complementary of X-ray macromolecular crystallography, and, as proven here, can confirm or deny previous results.