Kv1.3 current clamp assay developed on Qube 384 with Metrion Biosciences

Automated patch clamp experts: Stefano Stabilini, Senior Scientist from Metrion Biosciences, and our Sophion Application Scientist Beatrice Badone have collaborated to develop a novel current clamp assay on the Qube 384. By screening compounds against the Kv1.3 ion channel in current clamp mode, the assay allows the direct effects of the potassium channel’s modulation to be measured in terms of the changes in membrane voltage.

Dr. Eddy Stevens, Director of Drug Discovery at Metrion Biosciences posits that this membrane voltage measure, e.g. Resting Membrane Potential, RMP, is the direct translation of the channel current activity and a definition of how excitable the compound has made the cell membrane.

Thus, for Kv1.3 modulating compounds, this Qube current clamp assay will allow the drug discovery of Kv1.3 inhibitors. The inhibitor activity will functionally translate into a hyperpolarized, less excitable cell membrane in effector memory T-cells (TEM), which have been identified as driving over-activity in autoimmune diseases (such as psoriasis).

Read the full application report on the new Kv1.3 assay here

World first: High throughput ion channel recordings of isolated primary dorsal root ganglion (DRG) neurons on Yale University’s Qube 384

Researchers at Yale University have published groundbreaking work. Dr Reza Ghovanloo and colleagues in Prof. Stephen Waxman’s lab have taken highly prized DRG nociceptive neuronal recordings from the very low throughput manual patch clamp technique onto the Qube high-throughput automated patch clamp.

Their methods will revolutionize the quantity and quality of the data obtainable from these pain pathway neurons, providing invaluable insights into the physiology of pain sensation.

The authors sum it up better than we can:

“In this proof-of-concept study, we applied adaptations to an automated high-throughput electrophysiological platform to the study of DRG neurons, which provides a model of neuronal cell types that manifests a high degree of diversity, and is relevant to pain, a global unmet medical need. However, this approach is applicable for the study of other excitable cell types”.

“Our results demonstrate the feasibility of patch clamp analysis of freshly isolated neurons on a high-throughput platform [Qube 384 eds]. This approach allows a blinded, unbiased, simultaneous, high-throughput, and comprehensive VC [voltage clamp] investigation of freshly isolated neurons, immediately after tissue dissociation. Moreover, CC [current clamp] analysis can be carried out following VC study, on the same neuron, in a high-throughput mode. This approach provides a basis for the high-throughput physiological and pharmacological study of a variety of types of channels and receptors within multiple types of freshly isolated neurons”.

Congratulations to Reza Ghovanloo, Sidharth Tyagi, Peng Zhao, Emre Kiziltug, Mark Estacion, Sulayman D. Dib-Hajj and Stephen G. Waxman on this seminal publication.

Read the full paper here

Sophion technology helped Metrion Biosciences meet higher customer demands and growing assay complexity

Metrion Biosciences successfully met the challenge of growing complexity in ion channel research by adopting Sophion Bioscience’s automated QPatch II and Qube 384 patch clamp platforms. The move transformed Metrion’s research output, enabling it to take on more – and larger – projects and grow its customer base.

Together, Metrion and Sophion have forged a close relationship over the years. Sophion’s input on assay optimization, application, and technical instrument support and instrument service has helped Metrion maximize the in-laboratory benefits of automated patch clamping.

Click to download the full story here

Challenging CRAC channel assays recorded on Sophion’s platforms and without any use of seal enhancers

Calcium release-activated calcium (CRAC) channels play prominent roles, among others, in autoimmune diseases, metastatic breast cancer, diabetes, inflammatory bowel disease, as well as having the potential to prevent transplant rejection. Consequently, molecules that modulate the activity of the CRAC channel current (I_CRAC) are of much interest.

Fluoride is a no-go in assays investigating CRAC channels. To achieve good, high resistance (giga-Ohm) membrane seals, some APC manufacturers rely heavily on seal enhancers like fluoride. Therefore, it has been considered difficult to conduct CRAC channel assays on automated patch clamp systems. With Sophion Bioscience’s platforms, we ensure true gigaseal formation in your experiments without the need for fluoride.

If you would like to learn more about performing CRAC channel assays using Sophion’s QPatch or Qube 384 automated patch clamp systems, read more here

Large Molecules: New application report focus on Wnt signaling pathway activation

In a new application report, written together with Dr. Aamir Ahmed from Kings College London, UK, we obtained automated patch clamp (APC) recordings using the fragile and scarce Wnt proteins. Wnt 9B, 5A and 10B, were all shown to activate ion channel currents in PC3 cells.

Wingless-related integration site (Wnt) comprises a diverse family of secreted signaling proteins (350–400 amino acids, 35-45 kDa), which act as close-range signaling molecules. Wnt signal activation initiates a complex downstream signal cascade in eukaryotic cells and is critical in the development of many diseases, including cancer.

It was possible to obtain both manual and automated patch clamp recordings of fragile and scarce Wnt proteins after a thorough optimization of the protein handling.

We highlight aspects of the handling of Wnt proteins optimized for APC testing, which could also be applied to other large molecules (e.g. peptide toxins, nanobodies, antibodies).

You can find all relevant Wnt signaling (and other large molecules) publications and posters here

Large molecule characterization using automated patch clamp

Automated patch clamp solutions have been used for years to routinely research ion channels on large molecules. Being able to screen and characterizing large molecules on automated patch clamp is the key to ensure an efficient drug discovery process.

Today, more than 90% of all approved drugs are coming from research on small molecules, but large molecules research is rapidly rising in prominence. The importance of drug discovery already constitute the lion’s share of the top 10 selling drugs worldwide.

Large molecules have gained more attention due to their mode of action, often achieving greater target specificity and potency than small molecule drugs.

Learn more about the various classes of large molecules and ion channel research on our Qube and QPatch solutions here

Pharmacological evaluation of GABAA receptor subtypes on Qube 384

Ligand-gated experiments require many compound additions for incubation, stimulation, test drug and combinations thereof. Enabling that in 384-format tremendously enhances the throughput which is necessary to find the interesting new NAMs, PAMs or other types of compounds.

Here we report studies of three subtypes of GABAA channels using Qube 384 and with focus on:

• Short ligand exposure with repetitive stimulations with EC50 concentrations of GABA
• Effects of agonists, antagonists and modulators
• Cumulative and non-cumulative concentration-response relationships
• Characterizing the pharmacological properties of four cell lines expressing different GABAA subtypes

For the full application report, please see here.

Sophion Seminar in Japan

Friday 19th October we hosted the annual Sophion Seminar in Tokyo. More than 40 interested visitors were there to hear interesting talks and see the QPatch II for the first time in Japan.

QPatch II was presented to the audience and among others, Prof. Koichi Nakajo gave an educational lecture on “Stochiometry and function of potassium channel auxiliary subunits”.

Also, Juha Kammonen from Charles Rivers, UK gave a talk on how they at Charles River have successfully implemented the Qube into their ion channel drug discovery screening cascade, and he presented example data from both a high throughput screening campaign and a hit-to-lead project.

CMIC Pharma Science was hosting the meeting – the settings were fantastic for the reception party that followed the meeting.

Mechanism of action elucidated with Qube

Two papers were recently published mechanistic studies; in Inhibitory effects of cannabidiol on voltage-dependent sodium currents Xenon together with Simon Frasier University used temperature control on Qube to describe the potency and mechanism of cannabidiol binding to voltage-gated sodium channels. Interestingly the potency is higher at lower temperatures. In Selective Nav1.7 Antagonists with Long Residence Xenon together with Genentech describe the mechanism of several of their compounds in selectively inhibiting Nav1.7, which is relevant in relation to inflammatory and neuropathic pain. These compounds act very slowly, but Qube allows more than one hour long experiments with very good voltage control during the whole experiment or as the authors state:

“Appropriate filters for minimum seal resistance and minimum current size were applied, and series resistance was compensated >80% to yield high- quality sodium channel recordings on par with manual voltage clamp techniques”

Qube is designed for drug discovery

We asked one of our customers how they screen several hundred thousands of compounds with high fidelity electrophysiology?

“We let the Qube run our large screening campaigns unattended during the night so we can run and develop other assays on the same instruments during the day”

Juha Kammonen, Principal Scientist, Charles River Laboratories

How easy is it to use Qube?

At Sophion we believe that ease of use is important. When a customer spontaneously express clear appreciation of how easy it is to use our systems, we know we got it right:

“It was true that it’s really up and running after two days training. We are very pleased”

Maija Ivaska, Research Assistant, Orion Pharma

Ease of use does not come by itself with sophisticated electrophysiological recordings in automated patch clamp, but is embedded in the Sophion DNA.

Ask for a demo anywhere in the world to experience this yourself. We are so confident that you will be allowed to run your own experiments at the end of the first day….and that is not always the case with APC instruments.

With our instruments you can perform experiments with a very low risk of human error and large degree standardization, enabling easy learning, many operators and high laboratory efficiency.

Read more about Qube here.

Fast desensitizing ion channels recorded on Qube 384

Some ligand gated ion channels have a tendency to desensitize over time, making it difficult to establish a stable assay baseline. Rapid compound washout can counteract desensitization by reducing ligand exposure time to a minimum.

On Qube 384 the minimum washout is now less than 1 second (0.8 s), enabling stable assay for fast desensitizing ion channels.

With nAChRa1 as model, acetyl choline as agonist and tetracaine as test compound, a stable and high performance assay was established with:

• Ligand exposure time 0.8 s
• Whole-cell resistance of average 1.2 GΩ
• Overall success rate of 89%
• Very tight data – CV of ≤ 9% for control rows and columns
• Z’ > 0.70
• No desensitization observed

You can read the whole report here.

Temperature control on Qube 384 – pharmacological dependency of hERG reference compounds

Studies of temperature dependencies can be done efficiently and reliably on Qube 384. In this study we directly measure change in potency with change in temperature on four different compounds, emphasizing the importance of temperature when studying drug candidates.

See the new application report here.