High affinity acylating antagonists for muscarinic receptors

Abstract

The muscarinic antagonists pirenzepine and telenzepine were derivatized as alkylamino derivatives at a site on the molecules corresponding to a region of bulk tolerance in receptor binding. The distal primary amino groups were coupled to the cross-linking reagent meta-phenylene diisothiocyanate, resulting in two isothiocyanate derivatives that were found to inhibit muscarinic receptors irreversibly and in a dose-dependent fashion. Preincubation of rat forebrain membranes with an isothiocyanate derivative followed by radioligand binding using [3H]N-methylscopolamine diminished the Bmax value, but did not affect the Kd value. The receptor binding site was not restored upon repeated washing, indicating that irreversible inhibition had occurred. IC50 values for the irreversible inhibition at rat forebrain muscarinic receptors were 0.15 nM and 0.19 nM, for derivatives of pirenzepine and telenzepine, respectively. The isothiocyanate derivative of pirenzepine was non-selective as an irreversible muscarinic inhibitor, and the corresponding derivative prepared from telenzepine was 5-fold selective for forebrain (mainly m1) vs. heart (m2) muscarinic receptors.

Fig. 1

Synthesis of irreversible inhibitors of muscarinic receptors derived from pirenzepine, 1a, or telenzepine, 1b. The structures of m-DITC-PAC and m-DITC-PAC are 4a and 4b, respectively.

Fig. 2

Effect of washing on radioligand binding to m-DITC-PAC-treated membranes. Rat brain membranes were either left untreated (○) or were treated (●) with 10 μM m-DITC-PAC affinity label for 30 min at room temperature, then washed for the indicated number of times with 25 ml of sodium phosphate buffer, and incubated in fresh buffer, before being assayed for [³H]NMS binding. Data are means from triplicate determinations whose S.D. was less than 4% of the mean.

Fig. 3

Scatchard analysis of untreated rat brain membranes (●) and membranes treated with 0.05 μM (▲) and 0.5 μM (■) m-DITC-PAC. Membranes were incubated with affinity label for 60 min at room temperature, then were washed twice with large volumes of buffer before being assayed for [³H]NMS binding.

Fig. 4

Effect of varying concentrations of m-DITC-PAC on the density of muscarinic antagonist binding sites, indicated by B_max values. Membranes were incubated with the indicated concentration of the affinity label, m-DITC-PAC and washed extensively. The treated membranes were then assayed for [³H]NMS binding and subjected to Scatchard analysis. The resulting B_max is plotted as a function of the concentration of the affinity label.

Figure 5

Effect of varying concentrations of m-DITC-PAC (a) and m-DITC-TAC (b) on [³H]NMS binding in membranes from rat brain (■) and from rat heart (●). Membranes were treated with the indicated concentration of compound for 60 min at room temperature, washed extensively, and assayed for [³H]NMS binding using a single point determination. Each data point is the mean of three separate determinations whose standard deviation did not exceed 7% of the mean.