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. 2020 Aug 31;11(19):3130–3143. doi: 10.1021/acschemneuro.0c00477

Figure 4.

Figure 4

Simulations of three- and four-state ligand binding at a receptor. A) Scheme depicting a surmountable antagonist ligand (L) binding to the receptor (R), in competition with the agonist (A; DA). B) Scheme depicting induced-fit insurmountable antagonist ligand binding, where RL* represents an irreversibly bound antagonist ligand. C, D, E) Simulation of response recovery from antagonism by SWR-1-8 (C), SV-III-130 (D), and SWR-1-14 (E) at WT D2R, with 1 or 100 μM DA during the recovery phase. The response is assumed to be proportional to the fraction of the agonist-bound state; RA. F) Simulated recovery of WT D2R activation by DA after prolonged, 400-s antagonism with SV-III-130. G) Experimental recovery of WT D2R activation by DA after prolonged, 400-s antagonism with SV-III-130. H) Simulated curve-shift assay with SV-III-130 at WT D2R, plotting the RA fraction after 500 s simulation time for different concentrations of DA and SV-III-130, as indicated. I) Simulated concentration–response curves for SV-III-130 antagonism at WT and L94A mutant D2R. “Normalized response” corresponds to the RA fraction after 100 s of simulation time, in the presence of 100 nM DA and varying concentrations of SV-III-130, as indicated. The L94A mutant was simulated by removing the fourth state, RL*, from the model. J) Simulation of response recovery from antagonism by SV-III-130 at the L94A mutant D2R. As in I), the three-state model is employed, using kinetic data for SV-III-130 from the L94A mutant. Simulations of SWR-1-8 (C; kon(RL) = 340 M–1 s–1, koff(RL) = 0.017 s–1), SV-III-130 (D; kon(RL) = 860 M–1 s–1, koff(RL) = 0.007 s–1, kon(RL*) = 0.01 s–1), and SWR-1-14 (E; kon(RL) = 300 M–1 s–1, koff(RL) = 0.021 s–1) at WT D2R were conducted in the presence of 1 or 100 μM DA as agonist. kon(A) = 5 × 106 M–1 s–1 and koff(A) = 0.17 s–1 for all simulations at WT D2R. Parameters for the three-state model of SV-III-130 interaction at D2R L94A; kon(RL) = 890 M–1 s–1, koff(RL) = 0.007/s, kon(A) = 5 × 106 M–1 s–1, and koff(A) = 0.21/s. koff(A) = was adjusted as necessary to yield a Kd corresponding to the DA EC50 at D2R L94A.