User's opinion on FIDA kinetics

Kinetics

A key parameter in Drug Discovery is the affinity for a drug against its target. This is the case whether the drug is a small molecule, peptide or protein (antibody). The affinity is a measure of how strong the interaction is between the drug and its target. For a simple 1:1 interaction

(1) [A]t + [B]t <=> [AB]t

where the subscript t denotes the time of the measurement and the net rate of formation of [AB] is defined as

(2) d[AB]/dt = kon x [A]t x [B]t  - koff x [AB]t

Equilibrium is then defined as the time where the net rates are equal, i.e.

(3) kon x [A]eq x [B]eq = koff x [AB]eq

where the subscript eq denotes the time at which the forward and reverse rates are identical and therefore the total rate of the system is 0. The affinity parameter Kd is then defined as

(4)  koff/kon = [A]eq x [B]eq / [AB]eq

In recent years, there has been a strong interest in not only evaluating the affinity between a drug and its target, but also in evaluating the kinetics that define the affinity. The off-rate (koff) has been of particular interest in that a slow off rate, or inversely, a long residence time defined as 1/koff can have significant consequences for efficacy by prolonging the engagement of the target even after the drug has cleared the bloodstream. Conversely, a slow off rate can also lead to prolonged engagement of off-targets which may be a critical down-side to a drug.

With the renewed interest in the kinetics governing the affinity between a drug and its target, it’s pivotal that a research team has access to technologies that allow for the rate constants to be determined. There are a number of kinetics technologies available today, each of which has its pros and cons, and it’s often advantageous to be able to utilize at least a couple of these technologies to not only cover a wide spectrum of interactions but also to be able to validate one technology against another.

There are a number of kinetics technologies available today, each of which has its pros and cons, and it’s often advantageous to be able to utilize at least a couple of these technologies to not only cover a wide spectrum of interactions but also to be able to validate one technology against another.

Technologies for kinetics

Surface Plasmon Resonance (SPR)

The gold standard in the field of kinetics in drug discovery has long been considered to be Surface Plasmon Resonance (SPR). SPR utilizes surface immobilized molecules on a gold chip (i.e. the ligand) over which interaction partners (i.e. analytes) are added in a flow system. Any interactions between ligand and analyte results in a change of the refracted index which is proportional to the molecular weight of the analyte. This allows for the kon and koff to be evaluated by changing the concentration of the analyte for each injection. In addition, the koff can also be determined in a concentration independent manner by running buffer over a surface with a preformed complex. A significant advantage of SPR is the ability to evaluate interactions between binding partners spanning a large range of molecular weights from ~200 Da all the way up to several hundreds of kDa. While SPR is an incredibly sensitive and informative technology, if often requires a high level of knowledge from the operator in order to develop robust assays. In addition, most SPR set-ups include an irreversibly immobilized ligand, limiting the amount of interactions that can be evaluated, especially when a slow koff is encountered.

Biolayer Interferometry (BLI)

Another technology, related to SPR, is Biolayer Interferometry (BLI). BLI also utilizes an immobilized ligand but instead of a flow system this technology uses dip probes that are inserted into reaction wells containing different concentrations of analyte. Compared to SPR, BLI typically requires less operator knowledge to develop assays, however, BLI is not as sensitive as SPR and is not suitable for studying interactions with small molecules (<1000 Da). Similar to SPR, BLI does allow for the direct determination of off rates by dipping a probe with a preformed complex into buffer.

Fluorescence Polarization (FP)

In addition to SPR and BLI, which both require immobilized ligands, fluorescent plate-based methods like Fluorescence Polarization (FP) and time-resolved Foerster resonance energy transfer (TR-FRET) offer the capability of measuring kinetics in solution-based systems. Most modern plate-readers are capable of reading 96-, 384, and 1536-well plates in kinetics mode, which allows these assays to be run in high-throughput formats.

FP measures the change in polarized fluorescence emission of molecules upon binding to an interaction partner. A requirement for robust FP signals is that the fluorescently labeled molecule is significantly smaller than the interaction partner which limits the scope of interactions that can be evaluated. In contrast, TR-FRET does not have these requirements and can in principle be used to evaluate any interaction independent of molecular weight.

However, while FP only requires one of the interaction partners to be fluorescently active, TR-FRET requires both a donor and an acceptor fluorophore which is most often achieved by utilizing specifically developed donor and acceptor molecules that reversibly bind to the two interaction partners that are being evaluated. In addition, the FRET signal is strongly dependent upon the distance between donor and acceptor which can often be a limitation in TR-FRET assays, especially when using antibody-labeled donor and acceptors. Lastly, it’s worth mentioning that while most plate readers are now equipped to read in kinetics mode, in order to ensure fast and reliable mixing of the reagents, the plate reader usually needs to be equipped with dedicated mixers which often adds significant cost to purchasing the instrument. Despite the potential drawbacks of kinetics evaluation by FP and TR-FRET there is a significant upside to these technologies in terms of throughput and sample consumption.

FIDA Kinetics

As outlined above, there are several technologies available on the market today that allow for kinetics evaluation of drug-target interactions. These technologies all have their pros and cons and there is definitely a need for additional tools in the kinetics tool box. One such tool is Flow Induced Dispersion Analysis (FIDA). With the recent launch of Fidabio’s kinetics module, Fidabio now provides an exciting alternative to the technologies described above. With the already established utility of the FIDA platform in equilibrium mode, including affinity measurements, aggregation measurements, size determination, buffer scouting etc. the expansion to now include collection and analysis of kinetics data will provide an exciting new tool as either a standalone technology or to compliment already established kinetics technologies.

FIDA is a first principles method that determines the diffusivity of a molecule or complex which is inversely proportional to its hydrodynamic radius (see how FIDA works). For most interactions, one of the two interacting partners is labeled with a fluorescently labeled tag, although intrinsic fluorescence can also be utilized. In contrast to FP, there is no requirements for the labeled molecule to be smaller than the analyte.

The diffusivity of the labelled molecule can then be measured as a function of analyte concentration, and the affinity parameter, Kd, can be determined (read about FIDA kinetics). The two most common assay methods in the FIDA experiment is pre-mix, where the interaction partners are pre-mixed in a reaction well or vial prior to introduction into the capillary, and cap-mix where the two reactants are independently but simultaneously injected into the capillary.

In the cap-mix method, the mixing is performed in the capillary while in the pre-mix method the mixing is performed prior to capillary introduction. In both cases, the sample is then push forward in the capillary until it reaches the detector where the diffusivity of the mixture is measured.

The pressure, and thereby the speed of movement through the capillary, can be adjusted by the user, and it is this feature that allows the FIDA experiment to be run in kinetics mode. As described above, at equilibrium, the forward and reverse rates in a 1:1 interaction are identical. However, prior to that point, the forward rate will be faster than the reverse rate, which therefore, introduces time-dependency on the concentration of the formed complex. If the experiment is performed under pseudo first-order conditions, where one of the two reactants is in excess, the expression for the equilibrium rate constant is

(5) keq = kon x [B]T + koff

with [B]T denoting the total concentration of the excess reactant. In addition, the concentration of A at a given time can be described as

(6) [A]t = [A]eq + ([A]T - [A]eq]) x exp[-(kon x [B]T + koff) x t]

where [A]eq is the concentration of A when the system has reached equilibrium and [A]T and [B]T are the starting concentrations of A and B, respectively. When t < teq, the concentration of A is higher than what it will be at equilibrium, and conversely, the concentration of the complex AB is less than what it will be at equilibrium. By varying the mixing time in the capillary, the FIDA experiment can now be set up to measure the complex formation at several time points before equilibrium. Using the above expression, the software will allow for the analytical determination of both kon and koff. FIDA’s newly released kinetics software module will determine the kon and koff for a 1:1 model. However, the user also has the flexibility to export the time-dependent raw data and analyze these with more complex mechanisms if that are supported by the data.

FIDA’s newly released kinetics software module will determine the kon and koff for a 1:1 model. However, the user also has the flexibility to export the time-dependent raw data and analyze these with more complex mechanisms if that are supported by the data.

In conclusion, with the recent release of the FIDA kinetics module, there is now a new tool available to the researcher that will allow for the determination of the kinetics parameters of a given interaction in solution, with the additional benefits of the FIDA technology already established for the equilibrium experiment.

The Fidabio Team would like to thank Christian Kjaergaard, Ph.D., the author of this article, for his valuable insights and willingness to share them with a broader public.

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