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Identification of a Novel Allosteric Site at the M5 Muscarinic Acetylcholine Receptor
ACS Chemical Neuroscience ( IF 4.1 ) Pub Date : 2021-08-05 , DOI: 10.1021/acschemneuro.1c00383 Wessel A C Burger 1 , Patrick R Gentry 1 , Alice E Berizzi 1 , Ziva Vuckovic 1 , Emma T van der Westhuizen 1 , Geoff Thompson 1 , Mahmuda Yeasmin 1 , Craig W Lindsley 2, 3 , Patrick M Sexton 1, 4 , Christopher J Langmead 1 , Andrew B Tobin 5 , Arthur Christopoulos 1 , Celine Valant 1 , David M Thal 1
ACS Chemical Neuroscience ( IF 4.1 ) Pub Date : 2021-08-05 , DOI: 10.1021/acschemneuro.1c00383 Wessel A C Burger 1 , Patrick R Gentry 1 , Alice E Berizzi 1 , Ziva Vuckovic 1 , Emma T van der Westhuizen 1 , Geoff Thompson 1 , Mahmuda Yeasmin 1 , Craig W Lindsley 2, 3 , Patrick M Sexton 1, 4 , Christopher J Langmead 1 , Andrew B Tobin 5 , Arthur Christopoulos 1 , Celine Valant 1 , David M Thal 1
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The M5 muscarinic acetylcholine receptor (mAChR) has emerged as an exciting therapeutic target for the treatment of addiction and behavioral disorders. This has been in part due to promising preclinical studies with the M5 mAChR selective negative allosteric modulator (NAM), ML375. The binding site of ML375 remains unknown, however, making it difficult to develop improved M5 mAChR selective modulators. To determine the possible location of the ML375 binding site, we used radioligand binding and functional assays to show that ML375 does not interact with the well-characterized “common” mAChR allosteric site located in the receptor’s extracellular vestibule, nor a previously proposed second allosteric site recognized by the modulator, amiodarone. Molecular docking was used to predict potential allosteric sites within the transmembrane (TM) domain of the M5 mAChR. These predicted sites were assessed using M5–M2 mAChR receptor chimeras and further targeted with site-directed mutagenesis, which enabled the identification of a putative binding site for ML375 at the interface of TMs 2–4. Collectively, these results identify a third allosteric site at the M5 mAChR and highlight the ability of allosteric modulators to selectively target highly conserved proteins.
中文翻译：
M5 毒蕈碱乙酰胆碱受体的新型变构位点的鉴定
M 5毒蕈碱乙酰胆碱受体 (mAChR) 已成为治疗成瘾和行为障碍的令人兴奋的治疗靶点。这部分归功于 M 5 mAChR 选择性负变构调节剂 (NAM) ML375 的临床前研究。然而，ML375 的结合位点仍然未知，这使得开发改进的 M 5 mAChR 选择性调节剂变得困难。为了确定 ML375 结合位点的可能位置，我们使用放射性配体结合和功能测定来表明 ML375 不会与位于受体细胞外前庭的明确“常见”mAChR 变构位点相互作用，也不会与先前提出的第二个变构位点相互作用被调节剂胺碘酮识别。分子对接用于预测 M 5 mAChR 跨膜 (TM) 结构域内的潜在变构位点。使用 M 5 –M 2 mAChR 受体嵌合体评估这些预测位点，并进一步通过定点诱变进行靶向，从而能够在 TM 2-4 界面识别 ML375 的推定结合位点。总的来说，这些结果确定了 M 5 mAChR 上的第三个变构位点，并强调了变构调节剂选择性靶向高度保守蛋白质的能力。
更新日期：2021-08-19
中文翻译：
M5 毒蕈碱乙酰胆碱受体的新型变构位点的鉴定
M 5毒蕈碱乙酰胆碱受体 (mAChR) 已成为治疗成瘾和行为障碍的令人兴奋的治疗靶点。这部分归功于 M 5 mAChR 选择性负变构调节剂 (NAM) ML375 的临床前研究。然而，ML375 的结合位点仍然未知，这使得开发改进的 M 5 mAChR 选择性调节剂变得困难。为了确定 ML375 结合位点的可能位置，我们使用放射性配体结合和功能测定来表明 ML375 不会与位于受体细胞外前庭的明确“常见”mAChR 变构位点相互作用，也不会与先前提出的第二个变构位点相互作用被调节剂胺碘酮识别。分子对接用于预测 M 5 mAChR 跨膜 (TM) 结构域内的潜在变构位点。使用 M 5 –M 2 mAChR 受体嵌合体评估这些预测位点，并进一步通过定点诱变进行靶向，从而能够在 TM 2-4 界面识别 ML375 的推定结合位点。总的来说，这些结果确定了 M 5 mAChR 上的第三个变构位点，并强调了变构调节剂选择性靶向高度保守蛋白质的能力。
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