Ligand with two modes of interaction with the dopamine D2 receptor-an induced-fit mechanism of insurmountable antagonism

Kristoffer Sahlholm, Richard Agren, Hugo Zeberg, Tomasz Mac Iej Stepniewski, R. Benjamin Free, Sean W. Reilly, Robert R. Luedtke, Peter Arhem, Francisco Ciruela, David R. Sibley, Robert H. Mach, Jana Selent, Johanna Nilsson

Research output: Contribution to journalArticlepeer-review

Abstract

A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D2 receptor (D2R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D2R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 μM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 μM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of D2R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D2R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.

Original languageEnglish
Pages (from-to)3130-3143
Number of pages14
JournalACS Chemical Neuroscience
Volume11
Issue number19
DOIs
StatePublished - 7 Oct 2020

Keywords

  • Antipsychotics
  • Arrestin
  • Competitive binding
  • Drug kinetics
  • HEK cells
  • Molecular dynamics simulation
  • PET scan
  • Xenopus oocytes

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