Baicalin, a prodrug able to reach the CNS, is a prolyl oligopeptidase inhibitor

Abstract

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy terminus of proline residues. It has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore may have important clinical implications. In a previous work, we used ¹⁹F NMR to search for new prolyl oligopeptidase inhibitors from a library of traditional Chinese medicine plant extracts, and identified several extracts as powerful inhibitors of this peptidase. Here, the flavonoid baicalin was isolated as the active component of an extract of Scutellaria baicalensis roots having prolyl oligopeptidase inhibitory activity. Baicalin inhibited prolyl oligopeptidase in a dose-dependent manner. Inhibition experiments using baicalin analogs showed that the sugar moiety was not necessary for activity. The IC₅₀s of baicalin and its aglycone derivative baicalein were rather similar, showing that the sugar moiety was not involved in the interaction of baicalin with POP. These results were confirmed by saturation transfer difference NMR experiments. To further understand the absorption and transport mechanisms of baicalin and baicalein, we evaluated their transport in vitro through the gastrointestinal tract and the blood–brain barrier using a Parallel Artificial Membrane Permeability Assay. The molecule which potentially crosses both barriers was identified as baicalein, the aglycone moiety of baicalin. Our results show that baicalin is a new prodrug able to inhibit prolyl oligopeptidase. As baicalin is a natural compound with a long history of safe administration to humans, it is a highly attractive base from which to develop new treatments for schizophrenia, bipolar affective disorder, and related neuropsychiatric diseases.

Introduction

The protease prolyl oligopeptidase (POP; EC 3.4.21.26) is a cytosolic serine protease that hydrolyzes small proline-containing peptides at the carboxy terminus of proline-residues.1, 2 Many bioactive peptides such as substance P, thyroliberin, β-endorphin, and arginine–vasopressin are POP substrates.³ In our laboratory, POP was recently cloned from human brain RNA, expressed in Escherichia coli, and an homology model based on the X-ray structure of porcine POP was obtained.⁴

In recent years, POP has gained importance as a target for the treatment of schizophrenia (SZ), bipolar affective disorder (BD), and cognitive disturbances, such as those present in Alzheimer’s disease, mainly due to its involvement in the metabolism of inositol-1,4,5-P₃ (IP₃). IP₃ is a key molecule in the transduction cascade of neuropeptide signaling. Neuropeptides modulate levels of IP₃, which binds to its receptor in the membrane of the endoplasmic reticulum to induce the release of Ca²⁺, which is believed to play a crucial role in learning and memory.⁵ Recent findings have demonstrated that POP inhibition increases the concentration of IP₃.6, 7 The IP₃ signaling pathway participates in the therapeutic action of several mood-stabilizing drugs (lithium, carbamazepine, and valproic acid).⁸ Moreover, defects in the mechanisms that regulate IP₃ signaling may underlie BD, suggesting that other small-molecule inhibitors of POP may be useful in the treatment of the disease.⁹

In clinical studies, patients with BD or SZ exhibit abnormally high levels of serum POP activity.¹⁰ SZ affects 1% of the world’s population and has an enormous economic impact, as reflected by the fact that up to 90% of patients are unemployed. This impact is largely attributable to the lack of adequate therapeutic agents to treat the key cognitive symptoms; hence, a new class of drugs is required that addresses the cognitive deficits brought on by this disease.¹¹

POP inhibitors may prove valuable to treat various clinical conditions of the brain, as indicated by the neuroprotective and cognition-enhancing effects of POP inhibitors in experimental animals.12, 13, 14 The POP inhibitor S-17092-1 has been tested in Phase I trials for its capacity to enhance cognition.¹⁵ However, in spite of promising results, a drug based on POP inhibition has yet to reach the market.

An array of strategies is currently being used to identify POP inhibitors, including the exploration of natural products as a primary source of new inhibitory agents.16, 17, 18 Traditional Chinese medicine (TCM) dates back several thousands of years. China has 12,806 medicinal sources registered, including 11,145 plants.¹⁹ Moreover, 2375 compounds are compiled in the Pharmacopoeia of the People’s Republic of China (2000 edition).²⁰ Compounds with anti-cancer, anti-bacterial, anti-fungal, and anti-viral activities have been identified from these medicinal products.²¹

We recently reported a new POP enzymatic assay based on the combined use of ¹⁹F NMR and the fluorinated substrate ZGPF-4-CF₃. The assay is fast, reproducible, and circumvents the false positives and false negatives inherent to previously reported fluorimetric and colorimetric assays. It may therefore be useful for screening complex natural compound mixtures for new POP inhibitors.²² We later identified several extracts with powerful POP inhibitory activity, and isolated berberine as the POP inhibitory molecule present in Rhizoma coptidis extract.²³ In the present work, we fractionated the extract of Scutellaria baicalensis roots and identified baicalin as the POP inhibitory molecule. Inhibition studies using baicalin analogs and saturation transfer difference NMR experiments (STD) showed that the sugar moiety of baicalin was not necessary for POP inhibition. An in vitro study of the absorption pathways through the gastrointestinal tract and the blood–brain barrier (BBB) showed that the molecule which actually reaches the CNS is baicalein. Our results demonstrate that baicalin is a new prodrug able to inhibit POP.