Drug discovery is a critical and complex area of research. Rigorous testing, crucial safety considerations, and exhaustive data are all required for a drug candidate to progress into clinical trials. However, it is through this process that life-saving drugs such as Gleevec (a treatment for Chronic Myelogenous Leukemia with an incredible success rate) and Serelaxin (a novel treatment for heart failure) were discovered and made available.
In a drug discovery research workflow, potential drug candidates are often screened for their drug-receptor binding affinity using in-vitro techniques.1 This is done to predict the in-vivo efficacy of drug candidates, so that time and resources are spent productively during rigorous in-vivo testing. Inaccurate in-vitro measurements can waste months of work down the road. In a recent feature published in Drug Discovery Today, Dr. Sam R.J. Hoare shares a case study where such an event took place. Overlooking equilibrium time led to a poor prediction of in-vivo efficacy, ultimately leading to a loss of time and materials. However, by measuring full binding kinetics, the team at Neurocrine Biosciences was able to account for equilibrium time when predicting in-vivo efficacy and ultimately push two drug candidates through to the next stage of drug development.
In this blog, we dive into Dr. Hoare’s case study to share how using a quantitative binding kinetics technique such as surface plasmon resonance provides the data needed to accelerate your drug discovery pipeline.
Three important notes about binding affinity
In order to provide some context for this case study, there are three things you should know about binding affinity:
- Binding affinity is the concentration of a drug required to occupy 50% of the target molecules at equilibrium. This value quantifies the extent of target occupancy, which is used to predict in-vivo efficacy.1
- Residence time is a quantitative value representing the time a drug-receptor interaction takes to reach target occupancy. It is usually calculated as dissociation half time: t1/2 = 0.693/koff, where koff is the dissociation rate of the interaction (Hoare et al., 2019). An interaction is widely considered to be at equilibrium when five dissociation half times have passed.1
- In simplified terms, very slow dissociation (off) rates frequently result in erroneous affinity measurements due to not actually reaching equilibrium. This is just as critical when designing SPR experiments to measure steady-state affinity, but can be circumvented by measuring kinetics directly.
The Case Study
The following is a summary of the events outlined in Dr. Hoare’s case study. You can read the full publication here.
In 2003, the researchers at Neurocrine Biosciences were working to identify antagonists of the CRF1 (Corticotropin-releasing factor) receptor, a potential therapeutic target for psychiatric and endocrine disorders.1
Their top candidate, NBI30775, had done well in preclinical studies. However, it was discontinued for toxicological reasons.1 The binding affinity and in-vivo efficacy of NBI30775 were now being used as criteria for back-up candidates.
The team was now performing in-vivo testing of a new set of drug candidates. The selected candidates had shown promise in their 90-minute in-vitro competition assay, having a similar apparent affinity to that of NBI30775.1
The 5 lead candidates now had to show equivalent or greater efficacy compared to that of NBI30775 in an in-vivo biomarker assay. Unfortunately, despite the promising results in-vitro, all five candidates showed significantly lower efficacy than that of NBI30775. At this time, there was no obvious reason why.1
To investigate this phenomenon, the researchers titrated analogs of NBI30775 and one of the lead candidates, NBI35965.1 NBI30775 was found to be over ten times more potent in-vivo.1 This alarming observation led the team to perform radiolabeled binding kinetic studies of the analogs. Here’s what they found:
- The residence time (t1/2) of these compounds was very long – 5 hours for NBI35965 and 46 hours for NBI30775!1 Remember that a binding interaction is considered to be at equilibrium after five t1/2 periods. These compounds were clearly not at equilibrium when affinity was calculated in the 90-minute competition assay.
- When affinity was calculated from their binding kinetic rate values instead (the association rate, kon and dissociation rate, koff), the actual binding affinity of NBI30775 was ten times higher than that of NBI35965.1 This was primarily because NBI30775 had a much slower dissociation, leading to its long residence time.1
After assessing all five of their lead candidates in a binding kinetics assay, NBI30775 was found to have a much higher affinity and longer residence time than all of them.1 At this point, the team knew they had to change their screening criteria to better account for the long equilibrium time of their molecules.
The screening was now based on residence time: the candidates had to have a t1/2 time within one hour to that of NBI30775.1 Nine new candidates were selected based on this screening, and eight of these passed the original in-vivo efficacy criteria!1 This put the in-vivo efficacy prediction rate of their new in-vitro screening technique at 87%, an incredible improvement from their previous method. Of these eight candidates, two advanced through the next development, and one advanced into clinical trials.1
Why are binding kinetics important for accurate affinity measurements?
Binding kinetics are important because the time to reach equilibrium is dependent on the dissociation time.1 By definition, a binding interaction is at equilibrium when the opposing rates are equal. In this context, this is when association (kon) = dissociation (koff). Since association must occur first in order for dissociation to take place, equilibrium cannot be met until dissociation “catches up” with association. This is why residence time is calculated from the dissociation time and why full binding kinetics data is needed to accurately measure binding affinity, rather than measuring affinity indirectly. The published case study dives into this idea further, using simulations to measure exactly how affinity is distorted when equilibrium is overlooked.
This has important implications in a scenario like the one outlined in Dr. Hoare’s case study where the equilibrium time is very long.1 Equilibrium doesn’t have to be met in order to properly measure binding affinity, as affinity can be calculated from accurate kon and koff measurements. Therefore, measuring full binding kinetics is not only a more accurate way to calculate binding affinity, it can also be a much faster method as well.
Why are binding kinetics crucial in drug discovery?
As the authors write, the proper measurement of binding affinity has critical implications in drug discovery.1 If true affinity is higher than measured, patients could be overdosed during clinical trials. If true affinity is lower than measured, potentially life-saving drug candidates could be discontinued due to underdosing. In both cases, extensive time and resources could be wasted.
SPR provides the true affinity of drug-receptor interactions
The authors recommend several simple techniques to check for lack of equilibrium, such as an affinity-shift assay or the functional insurmountability test.1 However, to sufficiently screen drug candidates for clinical trials down the road, the authors recommend using a technique such as surface plasmon resonance to directly measuring binding kinetics.
Surface plasmon resonance (SPR) is a powerful technique for measuring the kon, koff, and overall affinity of drug-receptor interactions. By directly measuring the binding interaction in real-time without the use of labels, SPR provides reliable measurements to better predict the in-vivo efficacy of drug candidates. Unfortunately, researchers have previously had to choose alternative methods due to the cost and complexity of SPR. This was Nicoya’s inspiration behind creating OpenSPR™, an affordable and user-friendly surface plasmon resonance instrument. OpenSPR™ was specifically designed for ease-of-use and is priced in a range that is accessible to researchers. The results speak for themselves through the many publications from our OpenSPR™ users.
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- Hoare, S.R.J., Fleck, B.A., Williams, J.P., Grigoriadis, D.E. (2019). The importance of target binding kinetics for measuring target binding affinity in drug discovery: a case study from a CRF1 receptor antagonist program. Drug Discovery Today, 1-11. Doi:1016/j.drudis.2019.09.011