This study describes the localization and computational prediction of a putative binding site of an A₃ adenosine receptor (A₃AR) positive allosteric modulator LUF6000 (2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-(3,4-dichlorophenyl)amine). The work reveals an extrahelical lipid-facing binding pocket disparate from the orthosteric binding site that encompasses transmembrane domain (TMD) 1, TMD7, and Helix (H) 8, which was predicted by molecular modeling and validated by mutagenesis. According to the model, the nearly planar 1H-imidazo[4,5-c]quinolinamine ring system lies parallel to the transmembrane segments, inserted into an aromatic cage formed by - stacking interactions with the side chains of Y284^7.55 in TMD 7 and Y293^8.54 in H8, and by -NH bonding between Y284^7.55 and the exocyclic amine. The 2-cyclohexyl group is positioned 'upward' within a small hydrophobic sub-pocket created by residues in TMDs 1 and 7, while the 3,4-dichlorophenyl group extends toward the lipid interface. An H-bond between the N-1 amine of the heterocycle and the carbonyl of G29^1.49 further stabilizes the interaction. MD simulations predicted two metastable intermediates, one resembling a pose determined by molecular docking and a second involving transient interactions with Y293^8.54; in simulations, each of these intermediates converges into the final bound state. Structure-activity-relationships for replacement of either of the identified exocyclic or endocyclic amines with heteroatoms lacking H-bond donating ability were consistent with the hypothetical pose. Thus, we characterized an allosteric pocket for 1H-imidazo[4,5-c]quinolin-4-amines that is consistent with data generated by orthogonal methods, which will aid in the rational design of improved A₃AR PAMs.

Significance Statement Orthosteric A₃AR agonists have advanced in clinical trials for inflammatory conditions, liver diseases, and cancer. Thus, the clinical appeal of selective receptor activation could extend to allosteric enhancers, which would induce site- and time-specific activation in the affected tissue. By identifying the allosteric site for known positive allosteric modulators, structure-based drug discovery modalities can be enabled to enhance the pharmacological properties of the 1H-Imidazo[4,5-c]quinolin-4-amine class of A₃AR positive allosteric modulators.

本研究描述了 A ₃腺苷受体 (A ₃ AR) 正变构调节剂 LUF6000 (2-环己基-1 H -咪唑并[4,5-c]喹啉-4-(3)的假定结合位点的定位和计算预测,4-二氯苯基)胺)。这项工作揭示了一个与正位结合位点不同的螺旋外脂质结合袋，该结合位点包含跨膜结构域 (TMD) 1、TMD7 和螺旋 (H) 8，这是通过分子模型预测并通过诱变验证的。根据该模型，近平面的 1 H -咪唑并[4,5-c]喹啉胺环系统与跨膜片段平行，插入通过与TMD 7 中Y284 ^7.55的侧链堆积相互作用形成的芳香笼中， H8 中的Y293 ^8.54，并通过 Y284 ^7.55和环外胺之间的 -NH 键合。2-环己基位于“向上”，位于 TMD 1 和 7 中的残基形成的小疏水亚袋内， 而 3,4-二氯苯基则向脂质界面延伸。杂环的N - 1胺和G29 ^1.49的羰基之间的H-键进一步稳定了相互作用。MD 模拟预测了两种亚稳态中间体，一种类似于通过分子对接确定的姿势，另一种涉及与 Y293 ^8.54的瞬时相互作用；在模拟中，这些中间体中的每一个都会收敛到最终的束缚态。用缺乏氢键供给能力的杂原子取代已识别的环外或环内胺的结构-活性关系与假设的姿势一致。因此，我们表征了 1 H -咪唑并[4,5-c]喹啉-4-胺的变构口袋，该变构口袋与正交方法生成的数据一致，这将有助于改进的 A ₃ AR PAM 的合理设计。

意义陈述正位 A ₃ AR 激动剂在治疗炎症、肝脏疾病和癌症的临床试验中取得了进展。因此，选择性受体激活的临床吸引力可以扩展到变构增强剂，这将在受影响的组织中诱导位点和时间特异性激活。通过识别已知正变构调节剂的变构位点，基于结构的药物发现模式可以增强A ₃ AR 正变构调节剂 1 H -咪唑并[4,5-c]喹啉-4-胺类的药理学特性调制器。