Superiority of the Triple-Acting 5-HT6R/5-HT3R Antagonist and MAO-B Reversible Inhibitor PZ-1922 over 5-HT6R Antagonist Intepirdine in Alleviation of Cognitive Deficits in Rats

The multifactorial origin and neurochemistry of Alzheimer’s disease (AD) call for the development of multitarget treatment strategies. We report a first-in-class triple acting compound that targets serotonin type 6 and 3 receptors (5-HT-Rs) and monoamine oxidase type B (MAO-B) as an approach for treating AD. The key structural features required for MAO-B inhibition and 5-HT6R antagonism and interaction with 5-HT3R were determined using molecular dynamic simulations and cryo-electron microscopy, respectively. Bioavailable PZ-1922 reversed scopolamine-induced cognitive deficits in the novel object recognition test. Furthermore, it displayed superior pro-cognitive properties compared to intepirdine (a 5-HT6R antagonist) in the AD model, which involved intracerebroventricular injection of an oligomeric solution of amyloid-β peptide (oAβ) in the T-maze test in rats. PZ-1922, but not intepirdine, restored levels of biomarkers characteristic of the debilitating effects of oAβ. These data support the potential of a multitarget approach involving the joint modulation of 5-HT6R/5-HT3R/MAO-B in AD.

The reaction vial was heated at 90˚C for 5h upon microwave-assisted conditions.The obtained product was converted to HCl salt of secondary amine upon overnight treatment with 1M methanolic solution of HCl and subsequent filtration.

Selectivity screen of PZ-1922
Compound PZ-1922 was tested for its selectivity over structurally related proteins and offtarget receptors.
Inhibition activity for MAO-A was measured using human recombinant MAO-A (Sigma Aldrich M7316) by fluorometric method according the previously reported protocol. 1The assay was carried out in 96-well plate. 2 mL of appropriate concentration of PZ-1922 in DMSO were added to wells that contained 98 mL of enzyme dilution (0.53 U/mL) in phosphate buffer (50 mM, pH 7.4).After the 30 min of preincubation in room temperature 50 mL of the solution of 800 mM 10-Acetyl-3,7-dihydroxyphenoxazine (Cayman Chemical Company 10010469) and 4 U/mL horse radish peroxidase (HRP, Sigma Aldrich P6782) was added and enzymatic reaction was started by addition of 50 mL of 800 mM p-tyramine (Alfa Aesar A12220) solution.
The signal was measured after 1h (excitation at 570 nm and emission at 585 nm) using EnSpire® multimode plate reader (PerkinElmer Inc.).Clorgyline (1 μM) was tested as reference compound.
Binding assays for serotonin 5-HT1AR, 5-HT7R and D2R were performed according to the previously reported methods. 2 HEK293 cells stably expressing human 5-HT1A, 5-HT7b and D2L receptors (prepared with the of Lipofectamine 2000) were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and grown in Dulbecco's Modified Eagle's Medium containing 10% dialyzed fetal bovine serum and 500 mg/mL G418 sulfate.For membrane preparation, cells were cultured in 150 cm 2 flasks, grown to 90% confluence, washed twice with pre-warmed to 37 °C phosphate buffered saline (PBS) and centrifuged (200 x g) in PBS containing 0.1 mM EDTA and 1 mM dithiothreitol.Prior to membrane preparation, pellets were stored at -80 °C.
Cell pellets were thawed and homogenized in 10 vol of assay buffer using an Ultra Turrax tissue homogenizer and centrifuged twice at 35 000 x g for 15 min at 4 °C, with incubation for 15 min at 37 °C between the centrifugations.The composition of the assay buffers was experimentally selected to achieve the maximum signal window.The assays for 5-HT7R and D2Rs were carried out in a total volume of 200 mL in 96-well plates for 1 h at 37 °C.The process of equilibration was terminated by rapid filtration through Unifilter plates with a 96well cell harvester and radioactivity retained on the filters was quantified on a Microbeta plate reader (PerkinElmer, USA).For displacement studies the assay samples contained as radioligands (PerkinElmer, USA): 2.5 nM     bound to the receptor.The ligand is also represented as sticks.While the densities are equivalent for the piperazine ring (in the background) and the 1H-pyrrolo [3,2-c]quinoline system, a clear difference is observed for the 3-chlorobenzyl and 3-chlorobenzenesulfonyl moieties at the entrance of the binding site.The density is less defined and broken for PZ-1939, indicative of the flexibility of this cycle.We hypothesize that this absence of ordering could be due to the two additional oxygen atoms that prevent a correct capping of the protein loop C that would immobilize the ligand.with precursor/predominant product ion transitions for the analytes.The mass spectral Q1→Q3 transitions monitored for PZ-1922 were m/z 377.1→334.1;377.1→308.2and 377.1→209.2and for internal standard (IS) was 305.03→248.09.The peak widths of precursor and product ions were set to 0.7 full width half-height.Quantification was done via peak area ratio.

Pharmacokinetic study -calculation
The area under the mean plasma and brain concentration versus time curve (AUC0→t) was calculated from zero to the last concentration point using the linear trapezoidal rule as: where   is the concentration of the compound.
The area under the first-moment curve (AUMC0 ®t) was estimated by calculation of the total area under the first-moment curve: where   is the time of the last sampling.
Mean residence time (MRT) was calculated as: Total clearance (Cl) was calculated as: Volume of distribution at steady-state (Vss) was calculated as: (Eq. 5) The absolute bioavailability after intragastric administration was calculated as: where   and   .are iv and ig doses of PZ-1922, respectively.

Figure S- 1 .
Figure S-1.The QM and MD data analysis results were obtained for PZ-1922 and PZ-1939 in the 5-HT6R.(A) Illustration of the intramolecular interactions via the gradient isosurfaces for conformations isolated from the most populated MD cluster.The egg-shaped fields refer to delocalized electrons of aromatic rings and were visualized for gradient surface factor s = 0.1 a.u.(B) Change of the C1-N1-C2-C3 dihedral angle during the MD simulation.(C) The multivariate plot shows the dependence of the distance versus the σ-hole angle for the halogen bond with A4.57 for each trajectory frame, divided into 10 ns-long interval ranges, and the reduced form of the plot presents the medians of the geometric parameters calculated for given time ranges of the trajectory.

Figure S- 2 .Figure S- 3 .
Figure S-2.The QM and MD data analysis results were obtained for PZ-1922 and PZ-1771 in the MAO-B enzyme.(A) Illustration of the intramolecular interactions via the gradient isosurfaces for conformations isolated from the most populated MD cluster.The egg-shaped fields refer to delocalized electrons of aromatic rings and were visualized for gradient surface factor s = 0.1 a.u.(B) Change of the C1-N1-C2-C3 dihedral angle during the MD simulation.(C) The multivariate plot shows the dependence of the distance versus the σ-hole angle for the halogen bond with ring centroid of F103 (for PZ-1922), and P102 (for PZ-1771) for each trajectory frame, divided into 10 ns-long interval ranges, and the reduced form of the plot presents the medians of the geometric parameters calculated for given time ranges of the trajectory.

Figure S- 4 .
Figure S-4.Image analysis workflow and quality density maps of the m5-HT3AR in complex with PZ-1939.(A) Schematic of the image analysis workflow (B) A representative micrograph of the dataset.(C) Selected 2D class averages of the final particles set.(D) Side view of the final reconstruction.The sharpened 3D density map is colored according to the local resolution (FSC threshold of 0.143).(E) Heat map of the angular distribution of particle projections for the reconstruction.(F) Gold-standard FSC curves.The dotted line represents the 0.143 FSC threshold.(G) Densities of the reconstructions in surface representation overlaid with the structure.From left to right: densities of the β-sheets in the ECD, densities of the Cys-loop and the M2-M3 loop, densities of helices M1 and M2, densities of M3 and M4, densities of M2 at the level of L9′ (L260).

Figure S- 5 .
Figure S-5.Comparison of ligand densities for PZ-1922 and PZ-1939.The Cryo-EM reconstructions are represented side by side PZ-1922 (left, yellow) and PZ-1939 (right, red)   bound to the receptor.The ligand is also represented as sticks.While the densities are equivalent for the piperazine ring (in the background) and the 1H-pyrrolo[3,2-c]quinoline system, a clear difference is observed for the 3-chlorobenzyl and 3-chlorobenzenesulfonyl moieties at the entrance of the binding site.The density is less defined and broken for PZ-1939, indicative of the flexibility of this cycle.We hypothesize that this absence of ordering could be due to the two additional oxygen atoms that prevent a correct capping of the protein loop C that would immobilize the ligand.

Figure S- 6 .
Figure S-6.Comparison of ligand poses for PZ-1922 with palonosetron and PZ-1939.The panels depict the overlays of PZ-1922 with palonosetron (yellow and pink) or PZ-1922 with PZ-1939 (yellow and cyan).

Figure S- 7 .
Figure S-7.The impact in the hippocampus of the curative (A-C) and preventive (D-F) strategies with intepirdine (INTEP) and PZ-1922 on apoptotic processes induced by the icv injection of Aβ25-35 were evaluated by Western blot.Variations of pro-caspase 3 (Pro-casp 3, 35 kDa) (A,B,D,E) and clived caspase 3 (Casp 3, 19 kDa) (A,C,D,F) were evaluated in each group, normalized with the variations of β-tubulin (β-tub, 55 kDa) and expressed in percent of variations obtained in non-injected rats (Naive group).For experimental protocols see Figure 6A and Figure 6G.All data are presented as box & whiskers with Min to Max and Median with n = 8 for Naive-V and Aβ-V groups; and n = 6 for Aβ-INTEP and Aβ-PZ-1922 groups.One-way ANOVA followed by Dunnett's multiple comparison was performed (See Table S-5).* p< 0.05; *** p< 0.001 and **** p< 0.0001 vs Aβ25-35 group treated with vehicle (Aβ-V).

Figure S- 8 .
Figure S-8.Impact of curative strategy with intepirdine (INTEP) and PZ-1922 on Aβ25-35-induced toxicity.(A,B) Spatial short-term memory performance was determined in a T-maze test and was expressed as the ratio of the time spent in the initially closed arm (B) over the time spent in the previous arm (A).(C-F) Synaptic Integrity in the hippocampus was evaluated by Western blot.Variations of post-(PSD95, 95 kDa) (C,D) and pre-synaptic (SYN, 65 kDa) (E,F) markers in the whole hippocampus were evaluated in each group, normalized with the respective variations of β-tubulin (β-tub, 55 kDa) and expressed in percent of variations obtained in non-injected rats (Naive group).For experimental protocol see Figure 6A.All data are presented as box & whiskers with Min to Max and Median with n = 8 for Naive (N), Scrambled (Sc) and Aβ25-35 (Aβ) rats treated with vehicle (VEH); and n = 6 for N, Sc and Aβ rats treated with intepirdine (INTEP) or PZ-1922.Two-way ANOVA followed by Tukey's multiple comparison was performed (See Table S-6).* p< 0.05 and ** p< 0.01 vs. respective naive (N) group; + p< 0.05 and ++ p< 0.01 vs. respective scrambled (Sc) group; ø p< 0.05 and øø p< 0.01 vs selected group.

Figure S- 11 .
Figure S-11.Impact of preventive strategy with intepirdine (INTEP) and PZ-1922 on Aβ25-35-induced toxicity.(A,B) Spatial short-term memory performance was determined in a T-maze test and was expressed as the ratio of the time spent in the initially closed arm (B) over the time spent in the previous arm (A).(C-F) Synaptic Integrity in the hippocampus was evaluated by Western blot.Variations of post-(PSD95, 95 kDa) (C,D) and pre-synaptic (SYN, 65 kDa) (E,F) markers in the whole hippocampus were evaluated in each group, normalized with the respective variations of β-tubulin (β-tub, 55 kDa) and expressed in percent of variations obtained in non-injected rats (Naive group).For experimental protocol see Figure 6G.All data are presented as box & whiskers with Min to Max and Median with n = 8 for Naive (N), Scrambled (Sc) and Aβ25-35 (Aβ) rats treated with vehicle (VEH); and n = 6 for N, Sc and Aβ rats treated with intepirdine (INTEP) or PZ-1922.Two-way ANOVA followed by Tukey's multiple comparison was performed (See Table S-6).* p< 0.05 and ** p< 0.01 vs. respective naive (N) group; + p< 0.05 and ++ p< 0.01 vs. respective scrambled (Sc) group; ø p< 0.05 and øø p< 0.01 vs selected group.

Figure S- 12 .
Figure S-12.The impact in the hippocampus of the preventive strategy with intepirdine (INTEP) and PZ-1922 on Cdk5 activity (A-H) induced by the icv injection of Aβ25-35 were evaluated by Western blot.Levels of Cdk5 (30 kDa) (A,B); p35 (35 kDa) (C,D), p25 (25 kDa) (E,F) and the level ratio of p25 / p35 (G,H) were evaluated in each group, normalized with the variations of β-tubulin (β-tub, 55 kDa) and expressed in percent of variations obtained in non-injected rats (Naive group).For experimental protocol see Figure 6G.All data are presented as box & whiskers with Min to Max and Median with n = 8 for Naive (N), Scrambled (Sc) and Aβ25-35 (Aβ) rats treated with vehicle (VEH); and n = 6 for N, Sc and Aβ rats treated with intepirdine (INTEP) or PZ-1922.Two-way ANOVA followed by Tukey's multiple comparison was performed (See Table S-6).* p< 0.05 and ** p< 0.01 vs. respective naive (N) group; + p< 0.05 and ++ p< 0.01 vs respective scrambled (Sc) group; ø p< 0.05 and øø p< 0.01 vs selected group.

Table S - 1 .
Cryo-EM data collection, refinement and validation statistics.Resolution determined by Gold-Standard FSC threshold of 0.143 for sharpened masked map.
a b Resolution determined by FSC threshold of 0.5 for sharpened masked map.

Table S -
6. Statistical analysis of supplementary figures.