The interaction between compounds and their drug targets – particularly the binding kinetics – is an essential part of the identification and validation of new therapeutics and small-molecule drugs. But, despite the many technological advances in research and development, many challenges remain in using kinetic analysis for the early stages of drug discovery.
In this whitepaper, we aim to shed light on these challenges, and how the Creoptix® WAVEsystem tackles them using the inherent sensitivity of the Grating-Coupled Interferometry (GCI) detection principle and use of non-clog WAVEchips. The whitepaper describes the method by which the system has addressed these challenges, along with examples of how it has been successful in doing so. Taken together, these examples demonstrate the versatility of GCI and how it is revolutionizing the study of molecular interactions and drug discovery.
Drug discovery is the process by which new candidate therapeutics are discovered. While the modern process can be lengthy and complex, historically drug discovery was much simpler. It began with the identification of plant extracts with therapeutic properties. It wasn’t until the late 1800s that drug discovery and development became more scientific, leading to an increase in the number of compounds identified and tested. However, due to a lack of systematization, not many more were developed.
Advances in molecular biology and the understanding of genetic data fueled efforts to identify drugs with greater specificity. By the mid-1900s, the pharmaceutical industry was moving toward rational drug discovery, using computational techniques along with advances in molecular biology to design therapeutics. Continued technological advances in optical biosensors revolutionized drug discovery by adding much-needed kinetic measurements to the already lengthy drug discovery process – further facilitating the identification of promising drug candidates.
Why kinetic interaction analysis is critical to drug discovery
The interaction between compounds and their drug targets is an essential part of the identification and validation of new therapeutics and small-molecule drugs. Affinity – defined as the strength of the binding interaction – is determined by two components: the rate at which a binding interaction decays (kd) and the association rate (ka), which describes the rate at which molecules bind. Together, they define the affinity (KD) of an interaction (KD = kd/ka).
The primary focus of drug developers has been to identify drug-target interactions that have high affinity or tight binding interactions. While affinity is an important factor in the success of a drug compound, it does not provide complete, comprehensive information about the interaction. Accurate measurement of the association and dissociation rates of interactions is also extremely important because it reveals the time component of the interaction.
Interactions with the same affinity can have very different association (ka) and dissociation (kd) rates, as shown in the diagram below. Each interaction shown reaches the same affinity (KD) in very different ways.
Figure 1: Example of two interactions with the same affinity but different kinetics (kd and ka)
Fortunately, with technological advances in optical biosensors such as biolayer interferometry (BLI), surface plasmon resonance (SPR), and grating-coupled interferometry (GCI), scientists have been able to further dissect kinetic parameters and monitor binding and dissociation events in real time. As a result, kinetic parameters such as ka and kd can be resolved, providing deeper insight into such interactions. This type of information is critical to understanding the biological system being studied and can determine whether a compound advances to the next stage in the drug discovery process. In addition, there is increasing evidence that kinetic parameters may have a stronger correlation with drug efficacy than affinity alone.
Techniques for kinetic interaction analysis
Several biophysical techniques can be used to analyze the binding kinetics of molecular interactions. These include:
- Surface plasmon resonance (SPR) – a label-free optical sensing technique that detects molecular interactions occurring on a gold or metal surface. Molecular interactions are detected as refractive index changes within a localized evanescent field of the surface plasmon, which can be monitored as energy dips at a specific angle of incidence. Microfluidic channels are integrated into the system to allow for the control and manipulation of small volumes, enabling more accurate measurements. However, these intricate microvalves can be prone to clogging and require lengthy repairs.
- Biolayer interferometry (BLI) – an optical biosensor that measures changes in the interference pattern of light reflected from a biosensor tip to detect binding events between molecules. A biosensor tip is typically coated with a capture molecule and is dipped in a sample solution containing an analyte of interest. Because BLI has no integrated microfluidics, it is virtually clog-free and easy to use, but sensitivity and accuracy are limited.
- Grating-coupled interferometry (GCI) – a label-free optical sensing technique used for real-time kinetic measurements. In this technique, a sample and reference beam are coupled into an optical waveguide. The light travels throughout the waveguide, creating an evanescent wave that spans the entire length of the sensor surface. The binding of a molecule to the immobilized target on the sensor causes a light phase shift, which is read out interferometrically. The binding kinetics are analyzed by measuring these phase-shift signals in real time.1 The long light-to-sample interaction length of the waveguide provides intrinsically high signal-to-noise levels. In addition, the microfluidic chip used in the GCI biosensor has a unique design that allows ultra-fast transition times for reliable determination of fast off-rates.
Figure 2: GCI: The measurement and the reference beams are coupled into the waveguide. Interference happens within the waveguide and a high-resolution, time-dependent and robust phase shift signal is created.
Challenges in kinetic interaction analysis for drug discovery
Despite the many technological advances in research and development, many challenges remain in the early stages of drug discovery, particularly in the determination of kinetics for complex targets. These challenges include:
- Large drug targets in combination with small molecule inhibitors, which requires techniques with higher sensitivity.
- Ability to resolve fast off-rates, which are required for fragment- or small-molecule screening.
- Compatibility with challenging targets, such as membrane proteins, receptors, or complex protein targets.
- Ability to determine kinetics in biologically relevant matrix compounds. Some compounds are presented as strong potential candidates but lack activity in the appropriate biologically relevant matrix. Streamlining this process by combining kinetics and testing in the relevant medium allows researchers to focus on more promising candidates.
- Ability to identify false-positive compounds more quickly for non-specific or non-functional regions. With increasing numbers of compounds or biologics being screened at one time, eliminating false-positive hits early on can save time and allow researchers to focus on more promising candidates.
- Ability to work with compounds with limited activity. Innovative kinetics methods (such as waveRAPID® – described in detail later) are needed to resolve the kinetics more quickly in order to work with these compounds.
Highlighted examples of GCI for drug discovery
The Creoptix® WAVEsystem aims to tackle these challenges using the inherent sensitivity of the GCI detection principle, together with the use of the non-clog WAVEchips. Here, we describe how these challenges can be addressed with the WAVEsystem, along with examples of how it has been done. Taken together, these examples demonstrate the versatility of GCI and how it is revolutionizing the study of molecular interactions and drug discovery.
Large drug targets in combination with small-molecule inhibitors: Pushing the limit of target-to-analyte molecular weight ratio for kinetic analysis
Small molecules represent an important class of therapeutics in drug discovery, but their interactions are inherently difficult to study. This can be a bottleneck when studying large drug targets in combination with small molecule inhibitors. This is because optical biosensors detect the change in mass. Therefore, the smaller the analyte, the lower the response levels. This makes studying small molecules that are less than 500 Da in size more difficult, with small-molecule fragments even going below 200-300 Da.
To combat this, it is possible to saturate the surface with the protein drug target, thereby increasing the number of binding targets on the surface. However, over-saturation of the surface introduces additional artifacts, such as mass transport or steric hindrances, which can affect the results. Surface saturation can therefore be a double-edged sword. Because GCI detects signals from across the entire sensor, it has inherently increased sensitivity, allowing the user to explore binding interactions with a reduced number of ligands on the surface. It can be used to identify and validate large drug targets in combination with small-molecule inhibitors.
The following study demonstrates the interaction of a small molecule (297 Da) with an undisclosed large drug target of 110 kDa, kindly provided by Novartis (NIBR, Cambridge MA, USA). Traditionally, an interaction with such a large ratio of drug target to analyte could only be reliably measured by saturating the surface with the target, maximizing response levels. In this example, we show that the WAVEsystem can determine the kinetics of this ligand-analyte pair at a high low-density surface. Reliable kinetics were recorded for this target-analyte pair within a broad range of immobilization levels and responses show that the effective limit for the target-to-analyte molecular weight ratio can be pushed to >1,000:1.
Figure 3. Kinetic interactions of a large target and small molecule determined using differential surface densities. A high-capacity PCH-STA Creoptix WAVEchip (pre-coated with streptavidin) was used to capture the target to the desired surface density level. Cycles included 15-30 startup cycles of running buffer injections, followed by a 1:3 dilution series of the analyte (9 concentrations, starting with 10 μM) with one blank injection after 3 analyte injections. DMSO calibrations were performed at the end of the series. All the cycles were run at 80 μl/min and included 45s baseline, 60s association, and 60s dissociation. All cycles were performed in triplicates.
Fast off-rate resolution to reduce false positives
Thanks to advances in computational modeling and combinatorial chemistry, drug developers can now perform fragment-based drug discovery (FBDD). Since fragments are much smaller in size, they can have much faster on- and off-rates. As standard instrumentation often cannot measure the kinetics of fast off-rates, fragments are often ranked based on affinity rather than kinetic parameters. However, binding affinity analysis alone does not provide a complete picture of the kinetic interaction. This can lead to many false positives, adding unnecessary time and cost to the workflow.
The WAVEsystem can be used to accurately measure fast off-rate kinetics of weakly binding molecules. Thanks to a microfluidic chip design that accommodates transition times as fast as 150 ms, the reliable determination of fast off-rates is possible up to 10 s-1 – increasing the quality of early-stage hit selection. In the example below, the WAVEsystem was methylsulfonamide to carbonic anhydrase II. Sensorgrams show clear resolution of association and dissociation for interactions in the mM range.
Figure 4: (A) Sensorgram of methylsulfonamide (95.1 Da) binding to Carbonic Anhydrase II (29 kDa) which was immobilized by amine-coupling on a PCH WAVEchip at 8500 pg/mm2 on flow cell 1. Data was acquired at 40Hz with a flow rate of 200μl/min. (B) Zoom into the association phase (left) and dissociation phase (right) with the respective residual distribution plots below showing the high and clear resolution of the kinetics. (C) Kinetic data compared to published data 
A match for small molecules and flexible fibrils
The formation of Beta-amyloid plaques is a hallmark of Alzheimer’s Disease. Beta-amyloids are composed of unstructured and disruptive aggregations of fibrils, a protein normally responsible for the structural integrity of cells. To identify treatments for Alzheimer’s disease, researchers have been working to identify small molecules that are able to disrupt the formation of fibril aggregates. Small molecule inhibitors usually have fast association and dissociation rates that require innovative fluidics and detection methods to resolve.
Here, we demonstrate how the WAVEsystem can resolve the binding kinetics of a small molecule inhibitor, thioflavin (THT) with fibrils (in collaboration with the Mario Negri Institute, Italy). In this experiment, fibrils were immobilized on a PCZ WAVEchip® (zwitterionic surface) and 1:2 serial dilutions of THT were exposed to the surface. At first glance at the kinetics, it appears that there is no curvature in the association and dissociation phases of the interaction, reaching equilibrium (saturation) very quickly; however, on closer examination, we can see that association and dissociation curvature can be clearly resolved. Curvature is imperative for the determination of kinetics to properly fit data to the evaluation engine.
Figure 5: Results of kinetic interaction analysis between small molecules and fibrils, using GCI with the WAVEchip®. Self-assembled fibrils were immobilized via amine coupling on a 4PCZ WAVEchip® at a surface density of approximately 8,000 pg/mm2. The small molecule thioflavin (THT, 319 Da) was injected at four different concentrations (50 µM – 6.25 µM) for 30 seconds at 400 µl per minute. Raw data were double-referenced and globally fitted with a 1:1 binding model.
Non-clog microfluidics: Reliable analysis of challenging samples
The WAVEsystem also provides crude sample robustness that can normally only be achieved with plate-based assays. The innovative design of the patented microfluidic chip supports body fluids, pathogenic samples, harsh solvents, and large particles of up to 1,000 nm, enabling reliable kinetic analysis of even unconventional samples. The innovative fluidics design integrates microfluidics into a disposable chip rather than in the instrument itself, removing any microvalves near the chip. Blockages, if any, are localized to the disposable chip and can be replaced with fresh microfluidics within minutes.
Here we show two examples by which the WAVEsystem is compatible with unconventional applications. First, determining the kinetics of a nanobody binding to a G-protein-coupled receptor (GPCR). This is a breakthrough application as GPCRs have been previously considered undruggable targets due to the challenges of working with the protein. In the second example, we show how the workflow of identifying antibodies for diagnostic applications can be optimized with the use of high percentage serum in kinetics measurement.
Molecular interactions between nanobody and GPCR
GPCRs are a large class of integral membrane proteins that regulate a variety of physiological processes. Despite their importance, GPCRs are notoriously difficult to study because they become unstable after extraction from the cell membrane. Moreover, residual membrane debris after purification can make the immobilization process challenging. The no-clog WAVEchips allow researchers to identify and validate therapeutics against GPCRs both in solution (detergent-solubilized) and in membranes, where they can retain their native conformation.
In this example, the WAVEsystem was used to analyze an interaction involving GPCRs captured from crude cell membrane extracts using centrifugation-sonication. In this study, crude membrane extracts from Chinese hamster ovary cells (provided by Novartis) were analyzed. By shortening the typical multistep purification process, this method required significantly less material, reagents, and preparation time. This offers an alternative method for drug validation for membrane proteins. Here, we show that the kinetics of nanobodies binding to GPCRs can be resolved, providing significant implications for drug selection and validation in the early stages of drug discovery.
Figure 6: Results from analysis of GPCRs from crude membrane extracts using GCI. A) The Nt-AVI target GPCR (red), the Ct-AVI target GPCR (green), and the negative control Nt-AVI GPCR (blue) were injected onto a 4PCP-STA WAVEchip® at 10 µl per minute. B) Five concentrations (0.96 nM – 3 µM) of the anti-target-GPCR nanobody were then injected in duplicate for 180 seconds at 45 µl per minute.
Antibody profiling in serum
Label-free analysis of molecular interactions in complex biofluids such as serum provides valuable information for the development of diagnostic antibodies. However, these matrices often place significant stress on surface-based biosensors, as various components can cause microfluidics to clog. This can result in lengthy program delays while critical maintenance is performed. Thanks to its innovative disposable microfluidics, the WAVEsystem is highly resistant to clogging. This enables kinetic analysis in physiologically relevant matrices in the early stages of discovery, providing superior insight compared to traditional surface plasmon resonance (SPR) technologies.
In this example, two different antibodies were immobilized and their binding kinetics with antigen was analyzed (provided by ProteoMediX AG, Switzerland). The results show that, while both Antibody 1 and Antibody 2 showed a dose response binding to the antigen, only Antibody 1 was able to bind to the antigen in 90% serum. The results of this study demonstrate that the non-clog GCI system not only provides a binary answer regarding the functionality of an antibody in serum but can also provide complete kinetic profiling in the presence of a biologically relevant matrix. The non-clog feature accelerates drug discovery by allowing profiling in the relevant matrix earlier in the development process.
Figure 7: Results from analysis of two antibodies in buffer with and in 90% serum. Two different antibodies were immobilized on a 4PCP chip via amine coupling. The respective antigen was injected in either a buffer (PBS P+) or 90% human serum in a dilution series of eight concentrations ranging from 137 pM to 300 nM, followed by 1,200 seconds of dissociation. Raw data were double-referenced and globally fit with a 1:1 binding model.
The flexibility of four channels in combination with waveRAPID®
Running four channels simultaneously
In a typical kinetics experiment, an empty reference surface is used to account for drift, bulk refractive index jumps, and non-specific binding. The reference signal is subtracted from the interaction signal to remove these effects from the final sensorgram. However, with the rise in complex drug targets, there is a need for additional referencing schemes to identify false positives early in the discovery process.
The addition of a reference ligand of similar size and structure could provide information on non-specific binding and similar drift to the protein interaction channel. The Creoptix® WAVEchips are designed for such flexibility. The chips contain four parallel flow cells that can be used simultaneously. They can be freely configured on the four-channel WAVEdelta system, allowing up to three reference surfaces to be included during extensive drug screens.
The new waveRAPID® method: Kinetic characterization from one single well
In addition to the flexibility of channel configuration, we have developed an alternative approach to measuring kinetics: the waveRAPID® method. This approach streamlines kinetic characterization by using pulses of increasing duration of an analyte at a single concentration2. Instead of probing the molecular interaction in serial dilutions one at a time, the concentration is modulated directly on the surface with pulses of increasing duration.
The kinetic information obtained from a single waveRAPID® cycle is sufficient to calculate kinetic parameters, making labor-intensive serial dilution workflows a thing of the past. In addition, unstable proteins that can lose activity over the course of a lengthy multi-cycle kinetics experiment can now be studied. This method streamlines kinetic characterization of large libraries and maximizes the number of interactions that can be studied with unstable protein targets.
Figure 8. waveRAPID® kinetics consists of a single cycle with each analyte pulse applied for increasing duration. The time-dependent concentration function is shown at the top left, and the sensorgram response is shown at the lower left. As with a traditional kinetics approach, the association and dissociation constants can be obtained by applying c(t) and R(t) values to the equation at the bottom. waveRAPID® assays can be interpreted using only the dissociation portions of a sensorgram (blue lines at right).
Screening with waveRAPID® identifies fragment hits against chromatin reader proteins
Here, a fragment screen was performed using a proprietary Fragment Library from CLS (Concept Life Science). The library, which consists of 1,100 fragments, was screened for hits against BRPF1, a chromatin reader, using waveRAPID®. Hit selection was based on kinetic rate constant fitting errors, Rmax, and KD values. This screen was completed in 54 hours for full kinetic parameters. Consumables for this screen included 2 sensorchips, 1,100 pipette tips, 4 assay plates and 2.1 L of running buffer.
In the experimental design, all four channels were used and run in parallel. In addition to an empty reference channel and the protein target channel, two additional reference ligands were used to identify compounds that bind to a counter target or non-specifically to the denatured protein target. This four-channel parallel flow experimental design allows for a multiplexed assay to probe multiple protein surfaces simultaneously. waveRAPID® streamlines workflow and maximizes the number of compounds that can be analyzed during the limited window for unstable targets.
Figure 9. waveRAPID® output of the interaction with six of the 1,100 fragments. (a) Experimental design with setup on the four different channels of the WAVEdelta GCI. (b) Example of waveRAPID® sensorgrams (red) fitted to a one-to-one binding model using the Direct Kinetics engine of WAVEcontrol (black), and providing Rmax, ka (association rate), kd (dissociation rate) and Kd (dissociation constant). (c) Resulting waveRAPID® rate map showing kd vs ka for each fragment binder (coloured by kd). The resulting Kd ranges are highlighted with dotted lines.
Taken together, the examples presented demonstrate how the superior sensitivity of GCI, combined with non-clog disposable chips, enables applications that can be challenging for other biosensors. Due to the inherent sensitivity of the detection principle, GCI is ideally suited for small-molecule or fragment screening. In addition, the patented disposable chips provide the ability to test crude or sticky samples.
The WAVEsystem is also designed to optimize existing scientific workflows, and its four parallel flow channels provide additional experimental flexibility. When combined with Creoptix’s waveRAPID® kinetic measurement approach, it can optimize lengthy and labor-intensive workflows while still providing reliable kinetic data. From small molecules to traditionally difficult-to-analyze compounds, GCI with the WAVEsystem is a reliable choice for a variety of drug discovery applications and can revolutionize molecular interaction analysis.
To learn more about the drug discovery analysis capabilities available to you with GCI, please learn more about Creoptix WAVEsystem here.
- Myszka, David G. “Analysis of small-molecule interactions using Biacore S51 technology.” Analytical biochemistry 329.2 (2004): 316-323
- Kartal, Önder et al. “waveRAPID-A Robust Assay for High-Throughput Kinetic Screens with the Creoptix WAVEsystem.” SLAS discovery: advancing life sciences R & D, 26:995–1003 (2021)