Dr Patrick McIntyre, Senior Research Scientist at Charnwood Molecular is our lead specialist for biophysical techniques and in particular surface plasmon resonance (SPR)
In this article Patrick outlines the techniques and their uses plus, answers some commonly asked questions about biophysics and SPR.
For anyone unfamiliar with it, can you describe what biophysical techniques are?
Ultimately, biophysical techniques are all about detecting the binding of one molecule to another – in our case typically a small molecule to a protein. You don’t get information on whether any functional changes are happening in your system (as with biochemical assays) or whether any phenotypic changes are happening (as with cell-based assays) – it is all about binding.
They can be used as part of an integrated project alongside biochemical or cell biology-based assays to quickly return binding data to our DMPK and medicinal chemistry colleagues. However, biophysics can also be used for standalone projects to definitively measure and characterise the binding of one molecule to another – the affinity, kinetics, thermodynamics, etc…
Why and when would you recommend using a biophysical approach?
We would always recommend the use of biophysics in any drug discovery project – the binding data it can yield can be highly informative and helpful at any stage of a project. Biophysics can be an ideal starting point for a project – quickly and definitively identifying hits. It can be more time and cost effective than starting a project with a cell-based assay and can be more widely applicable than a biochemical assay.
It is also a viable option when virtual screening may not be appropriate if a less complex approach is sufficient. Biophysical techniques strip this back, asking just the one question: “does my compound bind to this protein?”.
What is the most widely used biophysical technique?
There are many different biophysical techniques and here at Charnwood Molecular we have experience in and access to all of the most widely used ones – surface plasmon resonance (SPR), fluorescence-based thermal shift assay (FTSA), isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) and x-ray diffraction (XRD).
Surface plasmon resonance (SPR) is well-known and well-regarded as a powerful and highly sensitive technique for the study of biomolecular interactions. Under certain conditions, SPR can measure both the affinity (KD) of the interaction and the kinetics (on-rate (ka) and off-rate (kd). These parameters are highly valuable at all stages of a drug discovery project and clearly demonstrate the benefits of the technique.
At Charnwood Molecular we use the Biacore 8K system for our SPR work. It is recognised as a world-leader for biophysical characterisation of molecular interactions.
Focusing on SPR – how aware are your clients of this technique?
There’s no doubt that SPR is a well-known technique in large pharma and biotech companies. They may have their own specialists and instrumentation or know that this is something they look for in a CRO.
However, I have observed that smaller or start-up companies have heard of SPR but are not aware of how it lies within the biophysical as opposed to biochemistry sphere and the benefits it brings. For smaller companies I believe it would offer time and cost savings when used on a relevant project.
What are the most frequently asked questions regarding SPR?
These are the questions that I most often discuss with clients when assessing if SPR is suitable for their project:
Which protein is SPR applicable to?If a protein can be purified then we can try to develop an SPR assay for it.
Is the target suitable?The only strict requirement for the protein target is that it must be stable in the SPR chip environment for the duration of the binding experiment.
How sensitive is it i.e. how weak a hit can it detect?In some cases hits can be detected up to the millimolar range.
Affinity values and kinetic data can be measured between the low nanomolar to the mid micromolar range
What information can we acquire from it?
The simplest data we can acquire is whether one molecule is interacting with another – e.g., in a spot-test fragment screen format.
More complex data can be measured – the binding affinity (KD) between two molecules and the kinetics of this binding (on-rate (ka) and off-rate (k¬d)) and the stoichiometry of the interaction.
How fast do I get the data?
SPR can be very high throughput – depending on the experimental set up we can measure dozens of affinity values per day.
What is the likelihood of success?
The success of any SPR project relies on the successful immobilisation of one of the binding partners of the target interaction. There are myriad different immobilisation strategies to increase the chances of success – from direct covalent coupling to the chip surface to indirect capture by biotinylated antibodies.
It is rare that we do not consider SPR to have at least some likelihood of success for a particular project but even in these circumstances there is likely to be another biophysical method that we would consider applicable instead.
Looking further ahead in a project’s lifecycle, what does SPR contribute to the overall drug discovery cycle?
I believe the real value of SPR and other biophysical techniques to be towards the start of a drug discovery project when we can quickly and relatively simply identify hit matter against a target. The higher throughput and lower costs of SPR and other biophysical techniques means we can conduct multiple iterations of the ‘design-make-test’ cycle to rapidly progress initial hits into lead-like compounds.
Another particular benefit of SPR is being able to measure the kinetics of an interaction, which other techniques don’t necessarily do. Knowledge of an interaction’s kinetics can provide novel ways of triaging similar affinity compounds and feeds into decision making as far down the drug discovery cycle as compound formulation/dosing schedules. Ranking compounds by their off-rate, for instance, can help boost the residency time of a drug on its target.
How does SPR and biophysics more widely fit into the Charnwood Molecular bioscience team?
Charnwood Molecular’s bioscience team (formerly Aurelia Bioscience) is particularly well known for its cell biology reputation and expertise. I see our (the biophysics team) role in this respect being to identify and characterise genuine hit matter to progress to our cell biology colleagues for further functional and phenotypic testing. They are a large team with expertise in a variety of areas including flow cytometry, western blotting, high-content and live-cell imaging. If we can pass them fully characterised hit compounds that we have high confidence in then we believe this gives any drug discovery project the greatest chance of success.
I think it’s of great benefit to our clients that SPR can be conducted in-house with the results then passed over to our chemistry, DMPK and cell-biology colleagues at the next bench or lab. The project continues in good hands, all the way from initial hit finding through to lead generation.