In-Solution Measurements of Binding Kinetics and Thermodynamics by nano Temperature Alteration Kinetics
- 1NanoTemper Technologies GmbH
Abstract
Binding kinetics are central to mechanism-driven drug discovery. Previously, we established Spectral Shift technology for the measurement of equilibrium binding affinity by detecting small shifts in the emission spectrum of a fluorescently labeled molecule upon binding to a ligand. While slow binding kinetics can be observed as a gradual change toward equilibrium after mixing the binding partners, faster interactions often have already reached equilibrium once the measurement is started. Here, we extend Spectral Shift to the measurement of fast binding kinetics through a method termed "nano Temperature Alteration Kinetics" (nanoTAK). A rapid temperature step perturbs the binding equilibrium of an enthalpically driven interaction, and the subsequent relaxation toward the new equilibrium is monitored in real time by recording the time-dependent Spectral Shift signal. The relaxation directly encodes the association rate kon and dissociation rate koff of the interacting molecules, which are extracted by fitting data of a full ligand dilution series to a global kinetic model. In addition, the fit also returns thermodynamic parameters of the interaction such as the binding enthalpy and entropy. We validate the approach across three tiers of molecular interactions. First, hybridization of a 10-mer DNA duplex serves as a model system with large binding enthalpy. Global fits match the data over three different temperature steps and confirm the accuracy of the derived fit model. Second, the kinase p38 with a set of six ATP-competitive and allosteric inhibitors demonstrates that the method extends to a classical protein-small molecule system with substantially smaller enthalpic changes, yielding consistent rate constants across compounds and temperatures and offering unique insights into their temperature dependence. Third, the degrader system consisting of VHL E3-ligase complex (VCB), PROTAC MZ1 and bromodomain 2 of BRD4 (BRD4(BD2)) illustrates the application to a higher-order system, revealing differences in the binding kinetics between the binary and ternary complex. The presented method can resolve interactions that re-equilibrate faster than within ~1 second, a timescale that can be difficult to access reliably with surface-based instruments. Binding kinetics can be measured both near room temperature, using a temperature step from 20 to 25 {degrees}C, but also, as an inherent feature of the approach, at physiologically relevant temperatures such as 37 {degrees}C.
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BibTeX
@misc{langer2026insolution,
title = {{In-Solution Measurements of Binding Kinetics and Thermodynamics by nano Temperature Alteration Kinetics}},
author = {A. Langer and C. Osseforth and P. Schramm and M. Mohamadi and N.-E. Guenther and O. Reinecke},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.22.760345},
url = {https://www.biorxiv.org/content/10.64898/2026.10.22.760345v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - In-Solution Measurements of Binding Kinetics and Thermodynamics by nano Temperature Alteration Kinetics
AU - A. Langer
AU - C. Osseforth
AU - P. Schramm
AU - M. Mohamadi
AU - N.-E. Guenther
AU - O. Reinecke
PY - 2026
DA - 2026/10/22
PB - bioRxiv
DO - 10.64898/2026.10.22.760345
UR - https://www.biorxiv.org/content/10.64898/2026.10.22.760345v1
AB - Binding kinetics are central to mechanism-driven drug discovery. Previously, we established Spectral Shift technology for the measurement of equilibrium binding affinity by detecting small shifts in the emission spectrum of a fluorescently labeled molecule upon binding to a ligand. While slow binding kinetics can be observed as a gradual change toward equilibrium after mixing the binding partners, faster interactions often have already reached equilibrium once the measurement is started. Here, we extend Spectral Shift to the measurement of fast binding kinetics through a method termed "nano Temperature Alteration Kinetics" (nanoTAK). A rapid temperature step perturbs the binding equilibrium of an enthalpically driven interaction, and the subsequent relaxation toward the new equilibrium is monitored in real time by recording the time-dependent Spectral Shift signal. The relaxation directly encodes the association rate kon and dissociation rate koff of the interacting molecules, which are extracted by fitting data of a full ligand dilution series to a global kinetic model. In addition, the fit also returns thermodynamic parameters of the interaction such as the binding enthalpy and entropy. We validate the approach across three tiers of molecular interactions. First, hybridization of a 10-mer DNA duplex serves as a model system with large binding enthalpy. Global fits match the data over three different temperature steps and confirm the accuracy of the derived fit model. Second, the kinase p38 with a set of six ATP-competitive and allosteric inhibitors demonstrates that the method extends to a classical protein-small molecule system with substantially smaller enthalpic changes, yielding consistent rate constants across compounds and temperatures and offering unique insights into their temperature dependence. Third, the degrader system consisting of VHL E3-ligase complex (VCB), PROTAC MZ1 and bromodomain 2 of BRD4 (BRD4(BD2)) illustrates the application to a higher-order system, revealing differences in the binding kinetics between the binary and ternary complex. The presented method can resolve interactions that re-equilibrate faster than within ~1 second, a timescale that can be difficult to access reliably with surface-based instruments. Binding kinetics can be measured both near room temperature, using a temperature step from 20 to 25 {degrees}C, but also, as an inherent feature of the approach, at physiologically relevant temperatures such as 37 {degrees}C.
ER -