Investigating the Unbinding of Muscarinic Antagonists from the Muscarinic 3 Receptor

Abstract

Patient symptom relief is often heavily influenced by the residence time of the inhibitor-target complex. For the human muscarinic receptor 3 (hMR3), tiotropium is a long-acting bronchodilator used in 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 the unbinding rates could help develop the next generation of antimuscarinic agents. Using our novel unbinding algorithm, we were able to investigate ligand dissociation from hMR3. The unbinding paths of tiotropium and two of its analogues, N-methylscopolamin and homatropine methylbromide, show a consistent qualitative mechanism and allow us to identify the structural bottleneck of the process. Furthermore, our machine learning-based analysis identified key roles of the ECL2/TM5 junction involved in the transition state. Additionally, our results point to relevant changes at the intracellular end of the TM6 helix leading to the ICL3 kinase domain, highlighting the closest residue L482. This residue is located right between two main protein binding sites involved in signal transduction for hMR3's activation and regulation. We also highlight key pharmacophores of tiotropium that play determining roles in the unbinding kinetics and could aid toward drug design and lead optimization.

Figure 1

Structures of the ligands investigated in this study: tiotropium (1), N-methylscopolamin (2), and homatropine methylbromide (3).

Figure 2

Left: (a) overlay of the structures from the unbinding path of ligand 1 through time starting from the bound state (BS, in red) toward the unbound state (US, in blue) passing through an approximated transition state (TS, in white). Right: stick representations of the three unbound ligands on their original BS (b, d, and f for ligands 1–3, respectively) and their TS (c, e, and g for ligands 1–3).

Figure 3

Relative feature importance (RFI, top) and relative accuracy drop (RAD, bottom) are shown for every interatomic distance between ligand 1 and hMR3 in the 3 Å data set. Distances are ordered and clustered by residue number. Residues with the top six distances (red symbols) are highlighted.

Figure 4

Average RAD (from MLP) and RFI (from GBDT) of the interatomic distances of the ligand 1 per protein residue for the 6 Å data set. In red, the top 6 residues detected by both approaches.

Figure 5

Relative feature importance (RFI) (from the GBDT model) and relative accuracy drop (RAD) (from the MLP model) values for each interatomic ligand–protein distance per residue in the ligand 1′s 3 Å + ECL2/TM5 data set. Marked in red are the top distances for each model. The most important residues for the ML models are highlighted.

Figure 6

Top: front (a) and top (b) views of the M3 receptor at the TS; the most relevant residues for the unbinding process found by the ML models are shown in sticks. The residues belonging to the ECL2 loop are shown in salmon, which is found to be the most relevant region. (c) Ligand 1′s structural representation with the most relevant atoms found by the MLTSA, highlighted, and annotated.

Figure 7

Panels (a–i) are the top nine residues represented as sticks with their protein–ligand (hMR3-ligand 1) distances consistently found to be the most important throughout the MLTSA analysis across all data sets. The ligand–protein complex at the TS and their distances are shown in yellow; the ones corresponding to the complex at the BS are shown in cyan. The atoms that the interatomic distances represented correspond to are represented as spheres.

Figure 8

Protein sequence alignment of hMR2 and hMR3 for selected regions involved in the unbinding process. Key residues identified by MLTSA are distinguished as conserved (cyan) or nonconserved (green) between the two receptors. The ECL2/TM5 region is also highlighted (purple and salmon).

Figure 9

(a) RFI and RAD for the allres (blue) and allres + wat (orange) data sets; highlighted are the top 5 residues for each approach (blue circle and orange diamond, respectively). (b) TS snapshot showing the top two water molecules as well as nearby residues as sticks in the allres + wat data set. The blue arrows highlight the displacement of the water molecules upon re-entering of ligand 1 in the binding site. (c) Diagram representation of the sequence of hMR3 portraying the different secondary structure motifs. In red, the top residues found decisive for the outcome by our MLTSA. In gray, the residues (kinase domain) not included in our simulation system. (d) Top important residues from MLTSA highlighted in the three-dimensional (3D) representation of hMR3, mostly corresponding to the ECL2/TM5 junction and the different ends of the α helices throughout the receptor.