Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats

Abstract

Recent studies suggest that subtype-selective activators of M(1)/M(4) muscarinic acetylcholine receptors (mAChRs) may offer a novel approach for the treatment of psychotic symptoms associated with schizophrenia and Alzheimer's disease. Previously developed muscarinic agonists have provided clinical data in support of this hypothesis, but failed in clinical development because of a lack of true subtype specificity and adverse effects associated with activation of other mAChR subtypes. We now report characterization of a novel highly selective agonist for the M(1) receptor with no agonist activity at any of the other mAChR subtypes, termed TBPB [1-(1'-2-methylbenzyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one]. Mutagenesis and molecular pharmacology studies revealed that TBPB activates M(1) through an allosteric site rather than the orthosteric acetylcholine binding site, which is likely critical for its unprecedented selectivity. Whole-cell patch-clamp recordings demonstrated that activation of M(1) by TBPB potentiates NMDA receptor currents in hippocampal pyramidal cells but does not alter excitatory or inhibitory synaptic transmission, responses thought to be mediated by M(2) and M(4). TBPB was efficacious in models predictive of antipsychotic-like activity in rats at doses that did not produce catalepsy or peripheral adverse effects of other mAChR agonists. Finally, TBPB had effects on the processing of the amyloid precursor protein toward the non-amyloidogenic pathway and decreased Abeta production in vitro. Together, these data suggest that selective activation of M(1) may provide a novel approach for the treatment of symptoms associated with schizophrenia and Alzheimer's disease.

Figure 1.

TBPB is an allosteric agonist of M₁ receptors in recombinant systems. TBPB (■; EC₅₀ of 158 ± 21 nm) and carbachol (▴; EC₅₀ of 28.6 ± 2.2 nm) elicit a robust response in CHO-K1 cells expressing WT rM₁. Data are mean ± SEM of three to six independent experiments each performed in quadruplicate.

Figure 2.

TBPB activity is consistent with action as an allosteric agonist in vitro. A, The Y381A mutation robustly shifts the M₁ response to CCh. WT rM₁ (■) EC₅₀ of 23.2 ± 2.2 nm. B, TBPB activates the Y381A mutant with an EC₅₀ similar to the WT rM₁. rM₁ (■) EC₅₀ of 220 ± 62 nm; Y381A (▴) EC₅₀ of 97.7 ± 36 nm. Data are mean ± SEM of five to six independent experiments each performed in quadruplicate.

Figure 3.

Atropine noncompetitively antagonizes TBPB at M₁. A, Increasing concentrations of atropine (Atr) (1–10 nm) competitively antagonize the action of CCh at the rM₁. B, Increasing concentrations of atropine (0.3–3.0 nm) produced a concentration-dependent decrease in the maximum effect of TBPB in CHO-K1 cells expressing WT rM₁. Data are mean ± SEM of three to six independent experiments each performed in triplicate. Veh, Vehicle.

Figure 4.

TBPB is a highly selective agonist for the M₁ receptor. A, Concentration–response curves for the orthosteric agonist AF267B across the M₁–M₅ mAChRs. rM₁ EC₅₀ of 149.7 nm (84.2% ACh Max), hM₂ EC₅₀ > 50 μm, hM₃ EC₅₀ of 38.2 nm (75.5% ACh Max), rM₄ EC₅₀ > 50 μm, hM₅ EC₅₀ of 1.08 μm (48.5% ACh Max). B, TBPB selectively activates the rM₁ subtype of muscarinic acetylcholine receptor. rM₁ (■) EC₅₀ of 112 ± 3.1 nm. Data are mean ± SEM of three independent experiments each performed in quadruplicate.

Figure 5.

TBPB regulates non-amyloidogenic APP processing. A, Western blots of conditioned media and cell lysates demonstrate increased production of APP_sα and CTFα in cells treated with CCh and TBPB. B, Quantitation of APP_sα band intensity shows a significant increase in APP_sα shedding from CCh- and TBPB-treated cells (*p < 0.01, n = 7 across 3 separate experiments for TBPB; **p < 0.001, n = 7 across 3 separate experiments for CCh). C, Quantitation of CTFα band intensity demonstrates a significant increase in production of CTFα in CCh- and TBPB-treated cells (**p < 0.001, n = 16 across 6 separate experiments). D, ELISA measurements from conditioned media show a significant decrease in secreted Aβ₄₀ from cells treated with either CCh or TBPB (**p < 0.001, n = 6 across 2 separate experiments for CCh; **p < 0.001, n = 9 across 3 separate experiments for TBPB).

Figure 6.

TBPB potentiates NMDA receptor currents in CA1 pyramidal cells. A, Representative traces of NMDA-evoked currents obtained before TBPB application (control), at the peak of TBPB-induced potentiation (3 μm TBPB) and after washout of TBPB. B, Time course studies reveal a slow development of TBPB-induced potentiation of NMDA receptor currents (*p < 0.05; **p < 0.01).

Figure 7.

TBPB had no effects on evoked EPSCs and IPSCs in CA1 hippocampal pyramidal cells. A, Average traces of EPSCs or IPSCs before application of TBPB or CCh (control), during application of TBPB (10 μm for EPSCs; 3 μm for IPSCs) or CCh (100 μm) from four different cells. TBPB had no effect on the amplitudes of evoked EPSCs (97.4 ± 4.0% of control; n = 5; p > 0.5) or IPSCs (106.5 ± 3.3% of control; n = 4; p > 0.1, Wilcoxon's matched-pair test) in hippocampal pyramidal cells. In parallel studies, CCh induced robust inhibition of EPSC and IPSC amplitudes (16.5 ± 4.0% of control, n = 5 and 15.4 ± 3.1%, n = 5, respectively; Wilcoxon's matched-pair test). B, Bar graph summarizes the effect of TBPB or CCh on EPSC and IPSC amplitude. All data represent mean ± SEM of five to six cells.

Figure 8.

TBPB elicits Fos expression in the rat forebrain. Representative photomicrographs illustrate that TBPB (100 mg/kg) induces Fos-like immunoreactivity in the PFC and shell region of the nucleus accumbens (NAS_shell) but not in the dorsolateral striatum (STR_dorsolateral). Scale bar, 50 μm

Figure 9.

TBPB produces a robust inhibition of amphetamine-induced hyperlocomotion. A, TBPB produces a dose-dependent decrease in amphetamine-induced hyperlocomotion. B, TBPB does not impair motor output at doses that produce antipsychotic-like activity. Data are expressed as mean ± SEM of the number of beam breaks/5 min. SEM are not shown if less than the size of the point (n = 6–8 per dose). *p < 0.05 vs vehicle plus amphetamine (1.0 mg/kg), Dunnett's test. Veh, Vehicle; Amph, amphetamine.

Figure 10.

TBPB produces antipsychotic-like effects at doses that do not produce catalepsy or induction of salivation. A, Dose- and time-related cataleptic immobility produced by haloperidol (middle) and lack of cataleptic immobility produced by TBPB (left) and clozapine (right) in rats. Haloperiodol produced catalepsy; significant after subcutaneous dose of 0.1 and 0.3 mg/kg by a Dunnett's comparison with the vehicle group. B, Dose-related induction of salivation produced by oxotremorine (left) and the lack of salivation produced by TBPB (right). Oxotremorine produced a robust dose-dependent induction of salivation, significant after subcutaneous dose of 0.1 and 0.3 mg/kg by a Dunnett's comparison with the vehicle (Veh) group. Results are expressed as mean ± SEM (n = 6 per treatment group). *p < 0.05 when compared with the vehicle control group.

Figure 11.

TBPB does not occupy central D₂ dopamine receptors at doses that produce antipsychotic-like effects. Representative PET images showing [¹⁸F]fallypride binding in rat striatum after vehicle (A–C) or a dose of TBPB (100 mg/kg, s.c.) (D), haloperidol (0.1 mg/kg, s.c.) (E), or haloperidol (1.0 mg/kg s.c.) (F) pretreatment. Single slices of the images that were summed over the duration of the scans are depicted. The slice thickness is 0.08 cm.