Table 6 Binding of K5, K17, K18 and MRS 1220 to the A₃R orthosteric binding area.

	E_vdW^a	E_EL^b	ΔG_solv^c	ΔG_eff^d	pK_B/pK_i^e
	E_vdW^a	E_EL^b	ΔG_solv^c	ΔG_eff^d	Schild analysis^f	NanoBRET^g	Radioligand binding^h
MRS 1220	– 64.6 ± 2.6	– 11.5 ± 2.5	39.2 ± 2.4	– 36.9 ± 3.6	10.07	9.99 ± 0.04	8.2–9.2
K5	– 42.0 ± 2.7	– 9.6 ± 5.2	30.8 ± 4.3	– 20.8 ± 4.3	ND	6.06 ± 0.09	5.02
K17	– 47.0 ± 2.4	– 8.8 ± 2.7	29.8 ± 2.9	– 25.9 ± 3.6	6.35	6.33 ± 0.03	5.38
K18	– 46.3 ± 2.9	– 7.5 ± 2.4	26.9 ± 3.1	– 26.9 ± 2.7	7.20	6.92 ± 0.10	6.05

Effective binding energies (ΔG_eff) and energy components (E_vdW, E_EL, ΔG_solv) in kcal mol⁻¹ calculated using the MM-PBSA method.
^avdW energy of binding calculated using molecular mechanics.
^bElectrostatic energy of binding calculated using molecular mechanics.
^cDifference in solvation energy between the complex, the protein and the ligand, i.e. G_{complex, solv}—(G_{protein, solv} + G_{ligand, solv}).
^dEffective binding free energy calculated as ΔG_eff = ΔE_ΜΜ + ΔG_sol; in Table 6, ΔE_ΜΜ = Ε_vdW ⁺ E_EL (see “Materials and methods”).
^eEquilibrium dissociation constant of MRS 1220, K5, K17 and K18 as determined through three independent experimental approaches: Schild analysis (pK_B), NanoBRET (pK_i) or radioligand binding (pK_i).
^fpK_B obtained through Schild analysis in A₃R stably expressing Flp-In CHO cells.
^gpK_i (mean ± SEM) obtained in NanoBRET binding assays using Nluc-A₃R stably expressing HEK 293 cells and determined through fitting our “Kinetics of competitive binding, rapid competitor dissociation” model or in the case of MRS 1220 through fitting with the ‘Kinetics of competitive binding’ model with a determined K_on (k₃) and K_off (k₄) rate of 3.25 ± 0.28 × 10⁸ M⁻¹ min⁻¹ and 0.0248 ± 0.005 min⁻¹, respectively.
^hpK_i values previously published for K5, K17 and K18 (Lagarias et al., 2018) or MRS 1220 (Stoddart et al., 2015) through radioligand binding assays.

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