Effects of Asparagine Mutagenesis of Conserved Aspartic Acids in Helix 2 (D2.50) and 3 (D3.32) of M₁–M₄ Muscarinic Receptors on the Irreversible Binding of Nitrogen Mustard Analogs of Acetylcholine and McN-A-343

Abstract

We investigated how asparagine mutagenesis of conserved aspartic acids in helix two (D2.50) and three (D3.32) of M1 – M4 muscarinic receptors alters the irreversible binding of acetylcholine mustard and BR384 (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate), a nitrogen mustard derivative of McN-A-343 ([4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl] trimethylammonium chloride). The D2.50N mutation moderately increased the affinity of the aziridinium ions of acetylcholine mustard and BR384 for M2 – M4 receptors and had little effect on the rate constant for receptor alkylation. The D3.32N mutation greatly reduced the rate constant for receptor alkylation by acetylcholine mustard, but not by BR384, although the affinity of BR384 was reduced. The combination of both mutations (D2.50N/D3.32N) substantially reduced the rate constant for receptor alkylation by BR384 relative to wild type and mutant D2.50N and D3.32N receptors. The change in binding affinity caused by the mutations suggests that the D2.50N mutation alters the interaction of acetylcholine mustard with D3.32 of M1 and M3 receptors, but not that of the M4 receptor. BR384 exhibited the converse relationship. The simplest explanation is that acetylcholine mustard and BR384 alkylate at least two residues on M1 – M4 receptors and that the D2.50N mutation alters the rate of alkylation of D3.32 relative to another residue, perhaps D2.50 itself.

Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor

“…This was also evident from an RMSD analysis of 23 and 28 (Figure S11, Supporting Information), although one has to keep in mind that the RMSD analysis considers the entire ligand structure. As reported previously for a MD simulation of the hM 2 R bound to compound 6, the highly conserved D103 3.32 , which typically interacts with MR agonists, 68 formed a cluster with S76 2.57 , W99 3.28 , ligand and receptor (yellow dashed lines) were identified for Y104 3.33 (o.) and N404 6.52 (o.)…”

“…This was also evident from an RMSD analysis of 23 and 28 (Figure S11, Supporting Information), although one has to keep in mind that the RMSD analysis considers the entire ligand structure. As reported previously for an MD simulation of the hM 2 R bound to compound 6 , the highly conserved D103 3.32 , which typically interacts with MR agonists, formed a cluster with S76 2.57 , W99 3.28 , Y426 7.39 , and Y430 7.43 (Figure S10A, Supporting Information) instead of interacting with the antagonists 23 and 28 . This was also reported for crystal structures of the M 1 R and M 4 R in complex with an antagonist …”

Harnessing Ion-Binding Sites for GPCR Pharmacology

“…For the muscarinic receptor subfamily, early studies suggested a classic Na 1 effect in M2 muscarinic receptors (Rosenberger et al, 1980), recently corroborated by mutation studies (Suga and Ehlert, 2013) and molecular dynamics simulations in M3 muscarinic receptors (Miao et al, 2015). However, a strong nonspecific effect of ionic strength (Birdsall et al, 1979) and ionic Harnessing Ion-Binding Sites for GPCR Pharmacology interactions in the orthosteric pocket may interfere with an accurate assessment of Na 1 selective binding in this subfamily.…”

Synthesis and biological comparison of enantiomers of mepenzolate bromide, a muscarinic receptor antagonist with bronchodilatory and anti-inflammatory activities

“…The enantiomers of mepenzolate, (R)-and (S)-mepenzolate ((R)-1 and (S)-1), were synthesized in two steps from commercially available benzilic acid (2) based on a procedure similar to that previously described 21 identified as Asp 3.32 (Asp148). This residue of these monoamine receptors strongly interacts with charged nitrogen atoms in the agonists and antagonists, 24 and subsequently we focused on this residue in hM 3 R (Asp148).…”