Therapeutic Effect of Novel Cyanopyrrolidine-Based Prolyl Oligopeptidase Inhibitors in Rat Models of Amnesia

Abstract

Prolyl oligopeptidase (POP) is a large cytosolic serine peptidase that is altered in patients with Alzheimer's disease, Parkinsonian syndrome, muscular dystrophies, and other denervating diseases. Thus, POP may represent a relevant therapeutic target for treatment of neuropsychiatric disorders and neurodegenerative diseases. Here, we report the characterization of five novel cyanopyrrolidine-based compounds (BocTrpPrdN, BocGlyPrdN, CbzMetPrdN, CbzGlnPrdN, and CbzAlaPrdN) and show that they are potent inhibitors of POP and are predicted to penetrate the blood-brain barrier (BBB). Indeed, we show that CbzMetPrdN penetrates the rat BBB and effectively inhibits POP in the brain when administered intraperitoneally. Furthermore, molecular modeling confirmed these compounds likely inhibit POP via interaction with the POP catalytic site. We evaluated protective effects of the cyanopyrrolidine-based POP inhibitors using scopolamine- and maximal electroshock-induced models of amnesia in rats and showed that BocTrpPrdN, BocGlyPrdN, CbzMetPrdN, and CbzGlnPrdN significantly prolonged conditioned passive avoidance reflex (CPAR) retention time when administered intraperitoneally (1 and 2 mg/kg) before evaluation in both models of amnesia, although CbzAlaPrdN was not effective in scopolamine-induced amnesia. Our data support previous reports on the antiamnesic effects of prolinal-based POP inhibitors and indicate an important role of POP in the regulation of learning and memory processes in the CNS.

Keywords: Alzheheimer’s disease; antiamnesic activity; blood brain barrier; brain; conditioned passive avoidance reflex; cyanopyrrolidine derivatives; prolyl oligopeptdases.

FIGURE 1

Structures of KYP-2047 and its novel analogs.

SCHEME 1

Synthesis of CbzMetPrdN. Reagents and conditions: a) PhSO₂Cl, in pyridine, 15–17°C, 16 h.

SCHEME 2

Synthesis of CbzGlnPrdN. Reagents and conditions: a) p-C₆H₆NO₂, DCC, in DMF, 0°C, 1 h; b) PhSO₂Cl, in pyridine, 10°C, 12 h; c) CF₃COOH, 30 min; d) Et₃N, in DMF, 16 h.

FIGURE 2

Docking pose of inhibitor KYP-2047 into the POP binding site (PDB code 3DDU). Residues within 3 Å of the pose are visible. H-bonds are shown as blue dashed lines.

FIGURE 3

Docking poses of CbzGlnPrdN (A) and CbzMetPrdN (B) into the POP binding site (PDB code 3DDU). Residues within 3 Å of the pose are visible. H-bonds are shown as blue dashed lines. The co-crystallized POP inhibitor from the 3DDU structure is depicted in thin magenta lines.

FIGURE 4

Time-dependent accumulation of CbzMetPrdN in brain tissue after a single i.p., administration (20 mg/kg) of the compound (n = 3 for each time point).

FIGURE 5

POP activity in brain tissue after a single i.p., administration (20 mg/kg) of CbzMetPrdN (n = 3 for each time point).

FIGURE 6

Effect of novel cyanopyrrolidine-based POP inhibitors on scopolamine-induced amnesia of conditioned passive avoidance reflex (CPAR). Rats in the group without treatment received scopolamine (1 mg/kg) 20 min before CPAR learning (green bar). Rats in the treatment groups received the indicated POP inhibitor (at doses 0.5, 1, or 2 mg/kg; white, gray, and black bars) or piracetam (300 mg/kg; blue bar) followed by a single injection of scopolamine (1 mg/kg) 20 min prior to CPAR learning. Rats in the negative control group received vehicle 40 min before CPAR learning (magenta bar). * Significant difference (p < 0.05) with nontreated rats (scopolamine only).

FIGURE 7

Effect of novel cyanopyrrolidine-based POP inhibitors on MES-induced amnesia of conditioned passive avoidance reflex (CPAR). Rats in the group without treatment received MES just after learning (green bar). Rats in the treatment groups received the indicated POP inhibitor (0.5, 1, or 2 mg/kg; white, gray, and black bars) or piracetam (300 mg/kg; blue bar) 40 min before CPAR learning, followed by MES just after learning. MES was induced using an electric shock generator (250 V, 120–122 20 mA, delivered for 0.1 s) by way of corneal electrodes. Rats in the negative control group received vehicle (magenta bar). * Significant difference (p < 0.05) with nontreated animals (MES only).