With a better understanding of the causes and mechanisms behind diseases arises a need for new treatment options and drugs to address them. However, since drug development is a lengthy and costly process with high attrition rates, the efficacies of potential drugs need to be determined as accurately and efficiently as possible. An important factor for efficacy is the strength of the interaction between a drug and its target, its binding affinity, which can be determined by kinetic binding experiments. Potential drugs must not only bind their targets, they also need to stay in place for a specific time in order to work efficiently.
Typically, screens determine the binding affinity of a compound at equilibrium at one timepoint, using different concentrations. This, however, neglects the plasticity of molecular interactions happening through continuous association (kon) and dissociation (koff), and with varying residence times. As these parameters impact on drug efficiency and the severity of side-effects, it is desirable to screen candidates for their kinetic parameters at an early time point.
While endpoint equilibrium experiments can easily be performed, the kinetic resolution of binding events has been a more complex endeavour, largely because assays were technically difficult and relatively low-throughput. A novel approach using TR-FRET in continuous read mode on a dedicated microplate reader is capable of simultaneously measuring the kinetics of hundreds of compounds. This offers the potential for placing binding kinetic analysis at the top of a screening cascade.
Kinetic binding assays: applying time-resolved FRET
Finding the right application for your needs is essential to assure high data quality and precise results. Since kinetic binding screenings are often performed in high-throughput, an important element is automation compatibility, as conversion of a benchtop to an automated high-throughput screening (HTS) assay enforces specific constraints affecting the practical assay design. Ideally, a HTS assay is performed in a single well, with a low amount of reagents (miniaturization), and minimal or no further manipulation than injection of the sample/compound to be tested. Accordingly, the choice of the optimal assay has to be subordinated to automation issues.
Establishing a stringent assay and an effective quality control method are major issues when setting up an automated screen. The clearer the distinction between negative and positive controls, the higher the possibility to obtain high-quality data with a neglectable number of false negative but especially false positive results. Specialised assays for the identification of false positives/negatives can also be employed for this purpose.
Homogeneous assays based on a simple mix and read principle, such as time-resolved Förster´s resonance energy transfer (TR-FRET), are particularly well-suited for kinetic binding screenings. In TR-FRET experiments, association and dissociation events are monitored by a shift in emission fluorescence which takes place, when a tracer molecule labelled with a donor and the target labelled with an acceptor fluorophore are in proximity to each other. By adding a potential drug that interferes with the tracer-target interaction this system is disrupted, resulting in a loss of TR-FRET signal (Figure 1).
Alternatively, labelled drugs can be employed to act as donors in a TR-FRET system (Figure 2). While this approach allows to directly assess drug-target association or dissociation events, it involves a substantial labelling effort when applied to whole compound libraries which should be taken into consideration.
Microplate reader requirements for kinetic binding screenings
BMG LATBECH´s PHERAstar® FSX is the only plate reader on the market able to kinetically resolve TR-FRET assays in high throughput. This high-end HTS plate reader is equipped with several key features dedicated to kinetic binding screenings, such as high temporal resolution, high-precision injectors with simultaneous reagent injection and detection, Simultaneous Dual Emission detection and full automation compatibility.
Speed and sensitivity are general requirements in drug screening to maximise the testing scale, and to keep overall costs low. Additionally, the ability to run TR-FRET detection in kinetic mode and a high temporal resolution in data acquisition are especially important in kinetic binding screenings, since drug binding events often occur in very narrow time windows, sometimes in a few seconds.
Together with the high temporal resolution, the capability to inject reagents and simultaneously acquire data, available on the PHERAstar FSX microplate reader, assures no down-time between drug addition and measurement. For TR-FRET assays, Simultaneous Dual Emission detection enables concomitant detection of donor and acceptor, halving read times and decreasing variability.
Finally, reliability is key, as errors occurring during a screen can result in repetitions, loss of time, reagents and consequently money. On the PHERAstar FSX, fast, precise, and stable measurements of low-volume samples of 10 µl and below enable the clear and dependable identification of hits from a vast number of compounds.
The market leading performance of the PHERAstar FSX ensures that binding events can be reliably identified and detected at low volumes and at the fastest speeds. This not only enables the highest throughput but also allows rapid cycle times in kinetic assays required for studying binding kinetics and on/off rates.
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