The dual inhibition of adenosine receptors A1 (A₁ AR) and A2 (A_2A AR) has been considered as an efficient strategy in the treatment of Parkinson’s disease (PD). This led to the recent development of a series of methoxy-substituted benzofuran derivatives among which compound 3j exhibited dual-inhibitory potencies in the micromolar range. Therefore, in this study, we seek to resolve the mechanisms by which this novel compound elicits its selective dual targeting against A₁ AR and A_2A AR. Unique to the binding of 3j in both proteins, from our findings, is the ring–ring interaction elicited by _A1Phe275 (→ _A2Phe170) with the benzofuran ring of the compound. As observed, this π-stacking interaction contributes notably to the stability of 3j at the active sites of A₁ and A_2A AR. Besides, conserved active site residues in the proteins such as _A1Ala170 (→ _A2Ala65), _A1Ile173 (→ _A2Ile68), _A1Val191 (→ _A2Val86), _A1Leu192 (→ _A2Leu87), _A1Ala195 (→ _A2Ala90), _A1Met284 (→ _A2Met179), _A1Tyr375 (→ _A2Tyr369), _A1Ile378 (→ _A2Ile372), and _A1His382 (→ _A2His376) were commonly involved with other ring substituents which further complement the dual binding and stability of 3j. This reflects a similar interaction mechanism that involved aromatic (π) interactions. Consequentially, vdW energies contributed immensely to the dual binding of the compound, which culminated in high ΔG_binds that were homogenous in both proteins. Furthermore, 3j commonly disrupted the stable and compact conformation of A₁ and A_2A AR, coupled with their active sites where Cα deviations were relatively high. Ligand mobility analysis also revealed that both compounds exhibited a similar motion pattern at the active site of the proteins relative to their optimal dual binding. We believe that findings from this study with significantly aid the structure-based design of highly selective dual-inhibitors of A₁ and A_2A AR.

腺苷受体A1（A ₁ AR）和A2（A _2A AR）的双重抑制被认为是治疗帕金森氏病（PD）的有效策略。这导致最近开发了一系列甲氧基取代的苯并呋喃衍生物，其中化合物3j在微摩尔范围内显示出双重抑制能力。因此，在这项研究中，我们寻求解决这种新颖化合物引发针对A ₁ AR和A _2A AR的选择性双重靶向的机制。根据我们的发现，两种蛋白质中3j的结合所特有的是_A1 Phe275（→ _A2Phe170）与苯并呋喃环的化合物。如所观察到的，这种π-堆叠相互作用显着地有助于3j在A ₁和A _2A AR的活性位点处的稳定性。此外，蛋白质中保守的活性位点残基如_A1 Ala170（→ _A2 Ala65），_A1 Ile173（→ _A2 Ile68），_A1 Val191（→ _A2 Val86），_A1 Leu192（→ _A2 Leu87），_A1 Ala195（→ _A2 Ala90） ，_A1 Met284（→ _A2 Met179），_A1 Tyr375（→ _A2 Tyr369），_A1 Ile378（→ _A2 Ile372）和_A1 His382（→ _A2 His376）通常与其他环取代基有关，它们进一步补充了3j的双重结合和稳定性。这反映出涉及芳香族（π）相互作用的类似相互作用机理。因此，vdW能量极大地促进了化合物的双重结合，最终达到了在两种蛋白质中均一的高ΔG_结合。此外，3j通常破坏A ₁和A _2A的稳定和紧凑构象AR及其Cα偏差相对较高的活性位点。配体迁移率分析还显示，相对于它们的最佳双重结合，两种化合物在蛋白质的活性位点均表现出相似的运动模式。我们认为，这项研究的结果大大有助于A ₁和A _2A AR高选择性双重抑制剂的基于结构的设计。