Webinar series: A rapid new way to measure kinetics

SelectScience^® was joined by an outstanding lineup of scientists from industry and academia for a successful virtual waveRAPID^® launch event featuring four informative sessions, produced in partnership with Creoptix. Experts in kinetics, Dr. Michael Hennig, Dr. Thomas Schubert, Dr. Nicolas Bocquet, Prof. Helena Danielson, and Dr. Kaspar Cottier shared how innovative technology can be used to study structural biology and biological interactions and explained the importance of kinetic measurements in the screening and discovery of novel drug molecules.

Read on for the Q&A highlights from the live sessions or watch the webinars on demand at a time that suits you.

Session 1: The new way of measuring kinetics

In the first session, Dr. Kaspar Cottier, CTO and Founder of Creoptix, explains why the measurement of kinetics is important when screening molecules and outlines the technology behind waveRAPID, how you can use the system, and the associated benefits.

How does rapid kinetics compare to traditional SPR?

KC: It’s quicker as you only need one injection, but result-wise you get the same result. You get the same kinetics, you get the on rates, you get the off rates, you get the affinity, but with only one injection, so it saves you time and material.

Can you explain a bit more about the sensitivity of this instrument compared to traditional SPR?

KC: Our GCI technology has a higher physical sensitivity. That means that the physical noise is lower than in traditional SPR. This is especially important for fast reactions where you do not have the time to do extensive averaging over several seconds as is the case with traditional SPR measurements.

Is there any limitation as to protein size or ligand size on this method?

KC: We have never run into a limitation. We have measured up to ratios between protein and compound of up to 1,000 to 1. That means that the target immobilized was 1,000 times larger or heavier than the analyte that is measured. This depends also on the activity that you get from your protein. If you have a challenging system, we will be happy to demonstrate it for you.

How long is a typical setup of a new project, for example, for membrane proteins?

KC: We routinely do end-customer demonstrations and complete measurements in less than a week, to get the first results. Then if you want to optimize the assay to get clean data in a screen, that might take you another few days. That’s the typical timeline that I would count on for the assay setup.

2: Use of waveRAPID^® method in drug discovery strategies for membrane proteins

In the second session, Dr. Nicolas Bocquet shows how leadXpro, an emerging lead compound discovery company, uses the WAVEsystem to assist its study of membrane protein structural biology. Bocquet also compares the use of the waveRAPID to classical methods for studying biologics and small molecules and illustrates the benefits of the waveRAPID method using screening examples.

Where do you see as the biggest advantages of GCI technology?

NB: The biggest advantage is the sensitivity. We're already working with small molecules and not usually super-active proteins and sensitivity is really key. Of course, all the developments with the speed and rapid are good because we can do much more work in the same amount of time.

What did the comparison of multiple cycle kinetics versus waveRAPID for biologics and small molecules mainly show?

NB: Before we jumped on waveRAPID, the comparison for us was to see the variations between the different measurements. For the nanobody, at least those we have measured and compared, the agreement was pretty high. That was just a confidence parameter that we can switch to waveRAPID more systematically and be confident about the data we get out of it.

For small molecules it's a bit trickier because you sometimes have more oddities in the shape, they misbehave more or we have problems in the solubility and detergent effects. The correlation is good but in our system it was still very high.

How could you combine primary and secondary screens with waveRAPID?

NB: Before waveRAPID, when we had a lot of compounds to screen, for example, we were doing one or two injections first to try to rank the binders. Then we were doing the full characterization of the best. Now with waveRAPID, we can process all of them at the same time. That's why I mentioned combining the primary and secondary screens. At the end of this run, you have the kinetic parameters already. Whereas before, you just had a vague estimation, and then you needed to do additional, more detailed screens to get the final parameters. waveRAPID is much faster in combining all the information in one run.

Have you ever tried to use a semi-purified GPCR untested small molecule, either agonist or antagonist binding?

NB: I have tried that. I could detect very small signals, but I have also tried it on soluble material, not even semi-purified, on full lysate cells. There, I could detect nanobody binding, it worked nicely. For small molecules, it's a bit more challenging as you need quite a good expression level, if you want to work with soluble material, it's a bit trickier. It depends on the system, but it can work if you have enough of the expressed protein in your lysate.

3: Rapid kinetics for fragments

In the third session, Prof. Helena Danielson, Professor of Biochemistry at Uppsala University in Sweden, explains how her team uses waveRAPID to resolve fragment kinetics and discusses the associated benefits of the system for drug discovery pipelines.

What challenges did the waveRAPID help you solve?

HD: I like the speed at which we can get new data, and that we have very good sensitivity when screening fragments. We can pick out hits well when we also account for kinetics. When we do fragment screening, it’s not trivial to see what a real hit is and what I would regard as just being something that gives a signal. When looking at the kinetic profile, more than just a square pulse gives us more confidence in the hits. It's building confidence in the hits we identify that would be a benefit in our case.

You observe the kinetics of fragments in waveRAPID, and not the Biacore system. Could you please explain that comparison in detail?

HD: When we use the Biacore, you're seeing how high the signal is and that’s not enough. You're always looking for a kinetic shape in addition to what we call the square pulse. Also, it's easier because we can get the information from the first screening data that we get. Normally with the Biacore experiment, we would do several series of experiments before selecting our hits. It’s simpler with the waveRAPID.

Were there any challenges on determining surface saturation?

HD: We always use reference compounds as controls. If you have good controls, there won't be any problems determining the surface saturation. If you have poor controls, you won't be quite sure of what the maximum signal would be at saturation.

Looking at the sensorgrams, SPR and Creoptix have very different kinetics. Could you take us through how you would decide which one is correct?

HD: If you look at simple square sensorgrams from a Biacore experiment, you know there's something interacting with the surface. Often you get strange sensorgrams for fragments, and they are often discarded as not being relevant. With Creoptix, we haven't seen those strange sorts of sensorgrams. Instead, we're seeing cleaner sensorgrams, and we're then picking them out on the basis of the kinetics. That means that we might also be selecting slightly different compounds depending on the different shape of the sensorgrams.

The simplest way to validate the hits that you are picking out is to repeat the experiment under exactly the same conditions, and maybe change the conditions as well to see whether that will give you similar sensorgrams. Increasing concentration can be one method, but it can also be a distraction if you have problems with solubility. Going to other techniques for a validation is useful, but I just recommend re-running experiments under different conditions.

4: Acceleration in drug discovery

In the final session of this webinar series, experts in kinetics, Dr. Michael Hennig, Dr. Thomas Schubert, Dr. Nicolas Bocquet, Prof. Helena Danielson, and Dr. Kaspar Cottier come together for a roundtable discussion on how this innovative technology can be used to study structural biology and biological interactions and the importance of kinetic measurements in the screening and discovery of novel drug molecules.

Would you say there's a growing need for kinetics information, specifically towards understanding mechanisms of action?

TS: Yes. Kinetics is powerful and important information that you need to understand what's going on between the target and the small molecule. Affinities alone will not give you sufficient information on that.

MH: The selection of the best compound for complex formation and structure determination, and this is independent of X-ray crystallography or electron microscopy, the best chance to get a complex and a good visualization of the compound is with a long residence time. We too check all our candidates for structure research with the Creoptix waveRAPID instrument, and then we prioritize our candidates for the structure determination.

HD: I think kinetics is the most important thing when we're working with biosensors. I wouldn't use a biosensor unless I get the kinetic information. It's the fundamental piece of information that I'm looking for. That’s why it has been frustrating working with fragments where you couldn't get the kinetics.

MH: An additional comment on binding kinetics and residence time. There has been a lot of literature around this. Recently, we had a very nice project where we could do a great color correlation between long residence time and finding disease-relevant cellular as well as in vivo activity. There are more and more examples in practice, company policies and the optimization of drug candidates — this is an important role of an exact measurement of the residence time and the cure rates.

What do you expect to see or hope to see for the future of this field? Do you see any trends that will be prevailing in the future?

HD: The initial focus on many of these technologies has been sensitivity, both in terms of how small molecules you can measure and the time resolution. Then throughput has been a feature that everybody has been looking for to speed up how we do things. I'm still waiting for more advanced ways of doing data analysis where we can extract more complex interactions, not just assuming that everything is a simple one-to-one interaction. Data analysis is something that I'm hoping for in the future.

TS: I agree. I would also emphasize the capability of HTS. Large screening approaches could be used in the future, and that could be the trend going forwards towards kinetic screening approaches.

KC: I also agree with Helena that data analysis is an important point that could be improved.

MH: Data analysis, to have good models in place for binding. What we have frequently is that the compounds stick to the proteins for unspecific binding. To differentiate between unspecific binding and specific binding would be a great move.

Find out more about waveRAPID^® and watch the webinars in the series on demand>>