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

Company

Prior PLM Medical, a company that specialises in supporting the medical and pharmaceutical industry to develop drug delivery devices from initial idea to end of product life.

Challenge

According to the World Health Organisation, over 300 million people worldwide suffer from respiratory diseases such as asthma and chronic obstructive pulmonary disorder (COPD). Inhaled medicine, typically in the form of pressurised metered dose inhalers (pMDI) and dry powder inhalers (DPI), is used to treat these diseases due to the direct delivery and reduced side effects. However, device/treatment efficacy is often quite poor with only 10-20% total lung deposition for most devices on the market.

The dynamics of the plume up to the point at which it exits the device are thought to influence speed and aerodynamic particle size distribution (APSD) and are considered important to drug transport to the lungs. However, this internal behaviour within the inhaler is not well understood due to the transient nature of the event and the difficulties in accessing the internal chambers within the device.

Sample

PMDI devices were investigated with a range of canisters, containing either HFA 134a or HFA 227ea propellants and various valve types, inserted into the actuator.

Solution

A monochromatic X-Ray beam was used at beamline ID19 at the European Synchrotron Radiation Facility. A Prior PLM Medical custom-built fixture was used to shake and actuate the inhalers. Phase contrast X-ray video of the dose release event from each inhaler showed the propellant mixture behaviour inside the canister and actuator. In addition mechanical interactions could be viewed taking place. This has provided new insights and is of value as a validation method for modelling efforts.

X-rays are capable of penetrating inhaler devices to visualise internal features but conventional techniques, for example industrial CT, are too slow to investigate fast events such as inhaler dose release. The ESRF beamlines have sufficient intensity to achieve the required temporal resolution and the phase contrast X-ray imaging technique gives excellent contrast for the low atomic number polymer/propellant/drug materials involved.

Benefits

“Our work at the ESRF has allowed us to see what’s happening inside both development stage and off-the-shelf commercial inhaler devices and has enabled our clients to make informed design decisions.  We also use the facility for our own internal R&D programmes and are very excited by the prospect of the ESRF Extremely Brilliant Source.” Alan McKiernan, Research Manager Prior PLM Medical, physicist.

 

High speed X-Ray video of the dose release from a commercial Dry Powder Inhaler from Prior PLM Medical on Vimeo.

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High speed phase contrast X-Ray imaging of dosing event in a pressurised Metered Dose Inhaler (left) and a Dry Powder Inhaler (right).

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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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.

 

 

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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.