Researchers at the University of Tubingen use Transcreener to uncover how residence time works on a molecular level with p38α MAPK Inhibitors – Many factors affect the effectiveness of ligand-substrate or drug-target interactions. Conformational docking and stearic considerations help inform optimal “fits.” Ligand moiety solubility, both in free and bound forms, is also a consideration due to the omnipresent influence of water. Once upon a time, association/dissociation measurements were all the rage. The lower the KD, the better. After all, if your ligand “sticks” more avidly to its target, shouldn’t that make it more effective? However, better docking does not necessarily mean longer docking. As rational drug design has evolved, total ligand residence time has been recognized as yet another factor in pharmacological efficacy.
Residence time is the time a drug remains bound to its target before dissociating; it is the reciprocal of dissociation rate (koff). For certain applications, remaining target-bound interactions for long periods, even after physiological clearance, can be advantageous.1,2
One commonly used method for measuring residence time is Surface Plasmon Residence (SPR), where proteins are bound to the surface of a chip and exposed to a stream of ligand. Jump dilution is an alternative method that measures the recovery of enzyme activity as the inhibitor dissociates.3
The Transcreener ADP2 FP (Fluorescence Polarization) assay can be used to measure residence times for kinase inhibitors by jump dilution because it allows continuous monitoring of ADP formation. In this method, the drug and target enzyme are incubated together to form the drug-target complex, and this complex is then diluted to dissociate. The ADP assay monitors the recovery of enzyme activity as the drug-target complex dissociates.4
MAPK14 Residence Time
p38-alpha MAPK (MAPK14) is a member of the MAPK family of proteins, critical for development and signaling under nominal conditions. MAPK14 plays an important role in response to inflammatory cytokines, cellular stress, and apoptosis. Its dysregulation has been implicated in inflammatory disorders, neurodegenerative disease, adverse cardiovascular remodeling, and cancer. While MAPK14 has been extensively studied and many inhibitors have been found, none have been approved for routine clinical use.5
Pantsar et al (2022) set out to explore the residence time parameters associated with molecular inhibitors of MAPK14. Understanding the factors affecting inhibitor residence time should enhance the drug optimization process.6
Utilizing various measures of IC50 and the two methods for measuring residence time, the authors assessed three different inhibitors with different structures and residence times. Two of the larger inhibitors (1 and 2) possessed similar low nM IC50 inhibition. A smaller, short-acting inhibitor was also profiled for comparison. Overall, their results showed that inhibitor residence time was enhanced by greater target conformational stability, less ligand solvent exposure, more buried ligand surface area, and a higher energy barrier to ligand resolvation.6 Focusing on differences in residence time measurements using the two methods, the authors assert that the jump dilution method with the Transcreener ADP assay may be more accurate than SPR because it measures inhibitor dissociation under more physiologically relevant biological conditions.6
Implications for Future Research
Finding clinically useful inhibitors of MAPK14 has been extraordinarily difficult. Appropriate MAPK14 modulation will likely be more useful for treatment than complete inhibition. In this case, precise measurement of the factors that affect MAPK14 residence time is crucial for designing new drugs.6
While SPR has been extensively used to measure residence time, it requires tethering proteins to a planar surface, which is likely to reduce their conformational freedom. On the other hand, the jump dilution method with the Transcreener ADP Assay allows ligands and targets to freely associate in solution and uses measurements of actual catalytic activity to determine functional residence time.3,4
1. Di Cera, E. (2020) Mechanisms of ligand binding. Biophys. Rev. 1(1): 011303. Review. https://doi.org/10.1063/5.0020997
2. Copeland, R. (2016) The drug-target residence time model: a 10-year retrospective. Nat Rev Drug Discov. 2016 Feb;15(2):87-95. https://doi.org/10.1038/nrd.2015.18.
3. Copeland RA. Drug–Target Residence Time. Evaluation of Enzyme Inhibitors in Drug Discovery: John Wiley & Sons, Inc.; 2013. p. 287-344.
4. Kumar, M. and Lowery, R.G. (2017) A High-Throughput Method for Measuring Drug Residence Time Using the Transcreener ADP Assay. SLAS Discovery, 22(7), 915-922. https://doi.org/10.1177/2472555217695080
5. Madkour, M.M. et al. (2021) Current status and future prospects of p38α/MAPK14 kinase and its inhibitors. European Journal of Medicinal Chemistry, 213, 113216. Review. https://doi.org/10.1016/j.ejmech.2021.113216
6. Pantsar, T. et al. (2022) Decisive role of water and protein dynamics in residence time of p38 alpha MAP kinase inhibitors. Nature Communications, 13:569. https://doi.org/10.1038/s41467-022-28164-4.