脯氨酰寡肽酶抑制剂的研究进展

目的综述脯氨酰寡肽酶抑制剂的构效关系、天然活性成分以及临床研究等方面的进展。方法根据国外相关文献 ,对脯氨酰寡肽酶抑制剂的构效关系、天然活性成分以及临床试验情况进行整理和归纳。结果脯氨酰寡肽酶抑制剂可通过设计合成或从天然药物的筛选中得到 ,数种口服抑制剂正在进行治疗Alzhermer’s病的临床试验。结论脯氨酰寡肽酶抑制剂有望成为新一代治疗Alzhermer’s病的重要药物     

脯氨酰寡肽酶功能及抑制剂研究进展

脯氨酰寡肽酶(prolyl oligopeptidase,POP)又称为脯氨酰内肽酶,是一种脯氨酸后切割酶,主要存在于细胞质中,负责短神经肽和肽激素的成熟和降解,与神经相关性疾病密切相关。POP的活性位点位于三联体催化结构域和β-螺旋桨结构域的界面。POP抑制剂具有神经保护、抗遗忘和增强认知的特性,同时对肿瘤、高血压等也有一定的治疗作用。本文就POP的功能、作用机制及其抑制剂的研究作一综述。     

Dicarboxylic acid bis(L-prolyl-pyrrolidine) amides as prolyl oligopeptidase inhibitors

New dicarboxylic acid bis(L-prolyl-pyrrolidine) amides were synthesized, and their inhibitory activity against prolyl oligopeptidase from pig brain was tested in vitro. As compared with earlier described prolyl oligopeptidase inhibitors, these new compounds have in common an L-prolyl-pyrrolidine moiety, but the typical lipophilic acyl end group is replaced by another L-prolyl-pyrrolidine moiety connected symmetrically with a short dicarboxylic acid linker. These compounds are a new type of peptidomimetic prolyl oligopeptidase inhibitor.     

Binding kinetics and duration of in vivo action of novel prolyl oligopeptidase inhibitors

Prolyl oligopeptidase (POP) is a serine protease that specifically hydrolyses small peptides at the carboxyl end of the proline residue. POP has gained pharmaceutical interest, since its inhibitors have been shown to have antiamnesic properties in rat. We examined the effect of the 2(S)-substituents CN and COCH(2)OH at the P1 site of the parent inhibitors isophthalic acid 2(S)-(cyclopentanecarbonyl)pyrrolidine-l-prolyl-pyrrolidine amide and 4-phenylbutanoyl-l-prolyl-pyrrolidine and bulky 5-t-butyl group at the P2 site l-prolyl residue of the parent inhibitor 4-phenylbutanoyl-l-prolyl-pyrrolidine on the binding kinetics to the enzyme. In addition, we studied the duration of POP inhibition in the rat tissues in vivo after i.p. administration. CN and COCH(2)OH substituents at the P1 site pyrrolidine group were found to greatly increase the affinity of the inhibitor and the enzyme-inhibitor complex half-life. In addition, 5-t-butyl group at the P2 site l-prolyl residue increased the dissociation half-life of the enzyme-inhibitor complex, without much affecting the inhibitory potency. The duration of the inhibition in the rat tissues followed the inhibition kinetic properties in that the compounds with fast dissociation produced shorter inhibition in the rat tissues than the compounds with slow dissociation. The duration of POP inhibition of compounds was evidently not governed by their serum clearance. The fact that the in vivo pharmacodynamic behaviour of POP inhibitors can be predicted by their in vitro-properties may be of importance when designing therapeutically useful POP inhibitors.     

New prolyl oligopeptidase inhibitors developed from dicarboxylic acid bis(l-prolyl-pyrrolidine) amides

Isophthalic acid bis(l-prolyl-pyrrolidine) amide is a very potent prolyl oligopeptidase inhibitor, but it has a log P value of -0.2, which is very low for a compound targeted to the brain. Therefore, these types of compounds were further modified to improve the structure-activity relationships, with the focus on increasing the log P value. The inhibitory activity against prolyl oligopeptidase from pig brain was tested in vitro. The most promising compounds resulted from replacing the pyrrolidinyl group at the P5 site by cycloalkyl groups, such as cyclopentyl and cyclohexyl groups, and by a phenyl group. These compounds are slightly more potent, and they have a significantly higher log P value. The potency of these compounds was further increased by replacing the pyrrolidinyl group at the P1 site by 2(S)-cyanopyrrolidinyl and 2(S)-(hydroxyacetyl)pyrrolidinyl groups.     

A cyclopent-2-enecarbonyl group mimics proline at the P2 position of prolyl oligopeptidase inhibitors

With the aim to replace the natural amino acid proline by a proline mimetic structure, a cyclopent-2-enecarbonyl moiety was studied at the P2 position of prolyl oligopeptidase (POP) inhibitors. The cyclopent-2-enecarbonyl moiety proved to be an excellent proline mimetic at the P2 position of POP inhibitors. The replacement is particularly useful when increased lipophilicity is needed.     

Brain pharmacokinetics of two prolyl oligopeptidase inhibitors, JTP-4819 and KYP-2047, in the rat

Abstract: Prolyl oligopeptidase (PREP) inhibitors are potential drug candidates for the treatment of neurological disorders, but little is known about their ability to cross the blood–brain barrier and to reach the target site. This study characterizes brain pharmacokinetics of two potent PREP inhibitors, JTP‐4819 and KYP‐2047. Firstly, the in vitro permeability (P_app) of JTP‐4819 and KYP‐2047 through a bovine brain microvessel endothelial cell monolayer was assessed. Then, the in vivo brain/blood ratio was determined for the total brain and plasma concentrations and also for the unbound extracellular drug concentrations after a single dose (50 μmol/kg i.p.). KYP‐2047 had a significantly higher P_app than JTP‐4819. In vivo, KYP‐2047 had higher total and unbound brain/blood ratios. KYP‐2047 was equally distributed between the cortex, hippocampus and striatum. In the case of JTP‐4819, the unbound brain extracellular concentrations could not be readily predicted from the unbound blood levels, probably because of its poor membrane penetration properties. KYP‐2047 displayed a better ability to reach the intracellularly located brain PREP, and it inhibited this enzyme more effectively than JTP‐4819 after an equimolar single dose. In conclusion, KYP‐2047 showed better brain penetration characteristics than JTP‐4819 both in vitro and in vivo. KYP‐2047 is a brain‐penetrating, potent and long‐acting PREP inhibitor; thus, it represents a convenient pharmacological tool for assessing the potential of PREP as a drug target.     

Dicarboxylic acid azacycle l-prolyl-pyrrolidine amides as prolyl oligopeptidase inhibitors and three-dimensional quantitative structure-activity relationship of the enzyme-inhibitor interactions

A series of dicarboxylic acid azacycle l-prolyl-pyrrolidine amides was synthesized, and their inhibitory activity against prolyl oligopeptidase (POP) from porcine brain was tested. Three different azacycles were tested at the position beyond P3 and six different dicarboxylic acids at the P3 position. l-Prolyl-pyrrolidine and l-prolyl-2(S)-cyanopyrrolidine were used at the P2-P1 positions. The IC(50) values ranged from 0.39 to 19000 nM. The most potent inhibitor was the 3,3-dimethylglutaric acid azepane l-prolyl-2(S)-cyanopyrrolidine amide. Molecular docking (GOLD) was used to analyze binding interactions between different POP inhibitors of this type and the POP enzyme. The data set consisted of the novel inhibitors, inhibitors published previously by our group, and well-known reference compounds. The alignments were further analyzed using comparative molecular similarity indices analysis. The binding of the inhibitors was consistent at the P1-P3 positions. Beyond the P3 position, two different binding modes were found, one that favors lipophilic structures and one that favors nonhydrophobic structures.     

Virtual screening and computational optimization for the discovery of covalent prolyl oligopeptidase inhibitors with activity in human cells

Our docking program, Fitted, implemented in our computational platform, Forecaster, has been modified to carry out automated virtual screening of covalent inhibitors. With this modified version of the program, virtual screening and further docking-based optimization of a selected hit led to the identification of potential covalent reversible inhibitors of prolyl oligopeptidase activity. After visual inspection, a virtual hit molecule together with four analogues were selected for synthesis and made in one-five chemical steps. Biological evaluations on recombinant POP and FAPα enzymes, cell extracts, and living cells demonstrated high potency and selectivity for POP over FAPα and DPPIV. Three compounds even exhibited high nanomolar inhibitory activities in intact living human cells and acceptable metabolic stability. This small set of molecules also demonstrated that covalent binding and/or geometrical constraints to the ligand/protein complex may lead to an increase in bioactivity.     

Pharmacogenomics and epilepsy: the road ahead

Epilepsy is one of the most common, serious neurological disorders, affecting an estimated 50 million people worldwide. The condition is typically treated using antiepileptic drugs of which there are 16 in widespread use. However, there are many different syndrome and seizure types within epilepsy and information guiding clinicians on the most effective drug and dose for individual patients is lacking. Further, all of the antiepileptic drugs have associated adverse reactions, some of which are severe and life-threatening. Here, we review the pharmacogenomic work to date in the context of these issues and comment on key aspects of study design that are required to speed up the identification of clinically relevant genetic factors.     

New fluorogenic substrates for the study of secondary specificity of prolyl oligopeptidase

The secondary specificity of prolyl oligopeptidase (POP) has been studied by using a series of fluorogenic substrates containing the highly fluorescent 7-amino-4-methyl-2-quinolinone (AMeq) marker. The substrates were dipeptides of the general formula Z-X-Pro-NH-Meq, bearing amino acid residues with variable functional groups [Met, Lys(Boc). Lys. His, Ser, Leu, Glu(OMe), Glu, Cys(Bzl)] at the P₂ position, and the tripeptide Z-Asn-Cys(Bzi)-Pro-NH-Meq. The kinetic parameters for their hydrolysis by porcine kidney POP were determined at λ_ex= 360 nm and λ_em= 430 nm. All the dipeptide substrates showed a high affinity to the enzyme and could be used for its fluorometric determination. The S₂ binding subsite of POP can accomodate amino acid residues with a bulky side group, while it prefers a positively charged group (free Lys) instead of a negatively charged one (free Glu). © Munksgaard 1997.     

Patent Evaluation: Bicyclic prolyl endopeptidase inhibitors

Summary

Novelty: Novel bicyclic nitrogen containing compounds, their enantiomers, diastereomers and epimers, are claimed. The compounds are neuroprotective and may be used to treat cognitive disorders, depression, anxiety and Alzheimer's disease.

Biology: Anti-prolyl-endopeptidase activity was determined according to the method of Yoshimoto and Tsuru (Agr. Biol. Chem. (1978) 42:2417) using Z-gly-Pro-p-nitroaniline as enzymatic substrate. IC₅₀ values were 3.8 × 10^-9 - 5 × 10^-8 M. Activity was measured in vitro and in vivo on rat cerebral cortex and IC₅₀ values were 1 × 10^-9 - 9 × 10^-8 M in vitro. In vivo inhibition was estimated at 35–65% with doses of 0.3–30 mg/kg.

Chemistry: Examples are provided for the syntheses of fifty-eight compounds by standard techniques and multistep reactions. Characterization is by mp, bp, optical rotation arid elemental analysis. The preferred compound is (2S,3aS,7aS)-1-[((1R,2R)-2-phenylcycloprop-1-yl)carbonyl]-2-[(1,3-thiazolidin-3-yl)carbonyl]perhydroindole.     

mTOR inhibitors: facing new challenges ahead

The enzyme mammalian target of rapamycin (mTOR) is a master kinase that regulates several critical intracellular processes. It is now well established that this enzyme has a key role in cancer and its inhibition as therapeutic anti-cancer strategy is well recognised. Several clinical trials using mTOR inhibitors have been and are currently being performed. A huge scientific literature exists not only reporting the results of these trials but also discussing the reasons for the limited efficacy of strategies used so far and the need for new strategies to overcome the problem of resistance. The aim of this review is mainly to reflect on how the complexity of the mTOR-dependent signalling pathway and our difficulty to untangle it can ultimately affect the development of proper strategies to fully exploit the potential benefits of mTOR inhibition as anti-cancer strategy.     

Beneficial effect of prolyl oligopeptidase inhibition on spatial memory in young but not in old scopolamine-treated rats

The effects of a novel prolyl oligopeptidase (POP) inhibitor KYP-2047 on spatial memory of young (3-month-old) and old (8- to 9-month-old) scopolamine-treated rats (0.4 mg/kg intraperitoneally) was investigated in the Morris water maze. In addition, the concentrations of promnesic neuropeptide substrates of POP, substance P and neurotensin in various brain areas after acute and chronic POP inhibition were measured in young rats. In addition, inositol-1,4,5-trisphosphate (IP(3)) levels were assayed in rat cortex and hippocampus after effective 2.5-day POP inhibition. KYP-2047 (1 or 5 mg/kg 30 min. before daily testing) dose-dependently improved the escape performance (i.e. latency to find the hidden platform and swimming path length) of the young but not the old rats in the water maze. POP inhibition had no consistent effect on substance P levels in cortex, hippocampus or hypothalamus, and only a modest increase in neurotensin concentration was observed in the hypothalamus after a single dose of KYP-2047. Moreover, IP(3) concentrations remained unaffected in cortex and hippocampus after POP inhibition. In conclusion, the behavioural data support the earlier findings of the promnesic action of POP inhibitors, but the mechanism of the memory-enhancing action remains unclear.     

Kinase inhibitors in cancer therapy: a look ahead

The most essential kinases involved in cell membrane receptor activation, signal transduction and cell cycle control or programmed cell death and their interconnections are reviewed. In tumours, the genes of many of those kinases are mutated or amplified or the proteins are overexpressed. The use of key kinases offers the possibility to screen in vitro for synthetic small molecule kinase inhibitors. In view of the many interconnections of cellular kinases, their role in preventing or inducing programmed cell death and the possibility that a considerable number of signal transducing proteins are still unknown, cellular test systems are recommended in which the respective key kinase or one of its main partner molecules are overexpressed.