Typically we run biochemical cellular assays that require a high concentration of molecules or cells to get the read out for the assays. With this kit we can watch a single molecule in action, following its binding to a receptor or to monitor its catalytic properties.LOL下注官网平台
This means that in future we can observe the action of a 单一 molecule rather than an ensemble of molecules. Combining the resulting data with the 阅读-outs of multiple molecule scans we can find 资讯rmation that would have been lost by measuring a collection of molecules alone.
It can screen and track molecules at concentrations that are a thousand-fold lower than previously (ultra-high sensitivity), and with that comes lower reagent consumption.
Not only can we extract synergy 资讯rmation but we’re also developing routines that allow us to extract kinetic 资讯rmation, such as how fast the molecules are interacting with the target and how fast they are leaving the target. This gives an idea of how long-lasting the effect of the molecule is.
The ultra-high sensitivity enables us to detect very weak interactions between the molecule and target. Most assays that for example include antibodies for detection usually depend on high-affinity binding, which means the molecule sits there for a long period of time. We can actually work equally well with the opposite, low-affinity binding, thereby significantly increasing measurement speed and providing much 更多 accurate 资讯rmation 关于 the binding kinetics. With this equipment, we don’t need to rely only on high-affinity antibodies when studying biomarkers in disease, and can additionally even define the bioactive fraction of a protein biomarker.
Stefan Geschwindner Principal Scientist, Discovery Sciences LOL下注网站
It enables us to do something we couldn’t do previously (detect a 单一 molecule’s activity). The cost of reagents is also reduced significantly due to 单一 molecule detection.
It provides data across different populations – we can detect heterogeneities and can see 资讯rmation that could be hidden in larger collections of molecules, so we can see how drugs act differently on particular forms of an enzyme or receptor that we wouldn’t be able to see in traditional assays. This could eventually provide an opportunity to further develop our personalised healthcare approach.
The project started around four years ago with the Chalmers University in Gothenburg using total internal reflection fluorescence or TIRF microscopy. Chalmers University still use the technology as well as other academic labs. We engaged with them to learn 关于 the technology and recognised that there was a huge opportunity for AstraZeneca.
It provides a unique opportunity to impact on our drug discovery efforts.
When you start a project you develop assays that allow chemists to view the outputs and understand the action. Most projects reach at some point a tight binding limit with high-affinity binding which means that a new assay needs to be developed. We think this platform could cover all levels of binding affinity from milli-molar right down to femto-molar levels.
In addition, the platform has been recently modified to generate the world-first automated TIRF microscope to meet todays´ screening requirements in drug discovery. This enables scientists to perform accurate kinetic measurements 更多 robustly, involving projects that have been originally deemed unfeasible to tackle with existing biophysical assay technologies.
We pitched the idea to invest in the microscope and received the equipment in 2015. For the first year we set out to show the impact of the technology in two of our projects. We exceeded this by impacting three different projects:
- Demonstrating ligand receptor selectivity – This technology has been successful in helping determine the exact affinity of a protein ligand to selected members of a receptor family in order to investigate if a mutated version of the ligand shows the desired receptor selectivity. Previous experiments employing traditional platforms did not display the required data quality and showed large variations. Experimenting with TIRF, we could validate a three-fold increase in selectivity to move the project forward
- Interaction of nuclear receptors with cofactors – Nuclear receptors can interact with an extensive range (>100) of cofactors when bound to drug molecules. These are essential to achieve the desired biological function and pharmacological response. Interaction profiles with these cofactors can be used to predict a drugs phamacological response. The plate-based nature of the TIRF platform has enabled us to generate profiles for all those cofactors simultaneously, where standard approaches can only generate information for a very limited number (<16) of cofactors or require very expensive setups
- Measuring kinetic binding data for unstable targets – Traditional surface-based approaches for the determination of kinetic binding data are frequently limited by the instability of the target protein when subjected to these investigations. The TIRF platform has enabled us to measure kinetic binding data for very unstable protein targets and to generate kinetic data of the desired quality to validate a critical project hypothesis. This work is part of a recent publication in Structure.
We have published six papers in the last two years, many in high-quality journals together with the academic supervisor. Several more are going through the peer-review process and recently our advances in technology developments have been positively highlighted by some key experts in a Nature Review publication.
A poster presentation at the Liposome Research Days won an award and was followed by an invitation to speak at a drug discovery conference and participate in a round-table discussion. As a result, the organisers have taken up 单一 molecule techniques as a theme for their next conference.
We are also excited by the enormous potential of single molecule techniques in late-stage drug discovery. We are looking at how we can apply this platform in late-stage discovery programmes, in particular with regard to biomarker detection which is currently being explored with the academic collaborator. A prospective view on the great potential of this technology for biosensing applications has been recently published in ACS Sensors.
Drug discovery at the single molecule level: inhibition-in-solution assay of membrane-reconstituted beta-secretase using single-molecule imagingLOL下注官网平台
Gunnarsson, A.; Snijder, A.; Hicks, J.; Gunnarsson, J.; Hook, F.; Geschwindner, S., Analytical Chemistry 2015, 87 (8), 4100-3.
Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid BilayerLOL下注官网平台
Gunnarsson, A.; Simonsson Nystrom, L.; Burazerovic, S.; Gunnarsson, J.; Snijder, A.; Geschwindner, S.; Hook, F., Chemistryopen 2016, 5 (5), 445-449.
Equilibrium-Fluctuation Analysis for Interaction Studies between Natural Ligands and Single G Protein-Coupled Receptors in Native Lipid VesiclesLOL下注官网平台
Wahlsten, O.; Gunnarsson, A.; Nystrom, L. S.; Pace, H.; Geschwindner, S.; Hook, F., Langmuir 2015, 31 (39), 10774-10780
Ligand Binding Mechanism in Steroid Receptors: From Conserved Plasticity to Differential Evolutionary ConstraintsLOL下注官网平台
Edman, K.; Hosseini, A.; Bjursell, M. K.; Aagaard, A.; Wissler, L.; Gunnarsson, A.; Kaminski, T.; Kohler, C.; Backstrom, S.; Jensen, T. J.; Cavallin, A.; Karlsson, U.; Nilsson, E.; Lecina, D.; Takahashi, R.; Grebner, C.; Geschwindner, S.; Lepisto, M.; Hogner, A. C.; Guallar, V., Structure 2015, 23 (12), 2280-229
Harnessing the Versatility of Optical Biosensors for Target-Based Small-Molecule Drug DiscoveryLOL下注官网平台
Kaminski, T.; Gunnarsson, A.; Geschwindner, S., ACS Sensors 2017, 2 (1), 10-15.
Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by OsteoarthritisLOL下注官网平台
Kohlhof, H.; Gravius, S.; Kohl, S.; Ahmad, S. S.; Randau, T.; Schmolders, J.; Rommelspacher, Y.; Friedrich, M.; Kaminski, T. P., Sci. Rep. 2016, 6.
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