Investigating the Unbinding of Muscarinic Antagonists from the Muscarinic 3 Receptor

Patient symptom relief is often heavily influenced bythe residencetime of the inhibitor-target complex. For the human muscarinicreceptor 3 (hMR3), tiotropium is a long-acting bronchodilator usedin conditions such as asthma or chronic obstructive pulmonary disease(COPD). The mechanistic insights into this inhibitor remain unclear;specifically, the elucidation of the main factors determining theunbinding rates could help develop the next generation of antimuscarinicagents. Using our novel unbinding algorithm, we were able to investigateligand dissociation from hMR3. The unbinding paths of tiotropium andtwo of its analogues, N-methylscopolamin and homatropinemethylbromide, show a consistent qualitative mechanism and allow usto identify the structural bottleneck of the process. Furthermore,our machine learning-based analysis identified key roles of the ECL2/TM5junction involved in the transition state. Additionally, our resultspoint to relevant changes at the intracellular end of the TM6 helixleading to the ICL3 kinase domain, highlighting the closest residueL482. This residue is located right between two main protein bindingsites involved in signal transduction for hMR3 & PRIME;s activationand regulation. We also highlight key pharmacophores of tiotropiumthat play determining roles in the unbinding kinetics and could aidtoward drug design and lead optimization.

Automated summary

For the human muscarinic receptor 3 (hMR3), the unbinding paths of tiotropium and its analogues were investigated, revealing a consistent qualitative mechanism and identifying the structural bottleneck. The machine learning-based analysis highlighted the important role of the ECL2/TM5 junction in the transition state. Additionally, relevant changes at the intracellular end of the TM6 helix leading to the ICL3 kinase domain were identified, emphasizing the significance of residue L482 in signal transduction.