Study On The Inhibitors Of GABA Shunt Enzymes

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. GABA is metabolized by the successive action of GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH), to succinic acid, which is a substrate for the tricarboxylic acid cycle. An increase in the brain GABA concentration can enhance the inhibitory function of GABAergic system and have therapeutic applications in neurological disorders including epilepsy, Parkinson's disease, Huntington's chorea, and Alzheimer's disease. Recently, it has been found that an increase in GABA also blocks the effects of drug addiction. An approach for increasing the brainGABA concentration would be to design a compound capable of permeating the blood-brain barrier that subsequently selectively inhibited GABA-T. Thus GABA-T has been validated as a target for neuroactive drugs.In this dissertation, it was established that 4-hydroxybenzaldehyde may serve as a lead structure for GABA-T and SSADH inhibitors. Based on the results, both 4-hydroxybenzaldehyde analogues were synthesized and evaluated for irreversible inhibition of GABA-T. The inhibitory effect of flavonoids on GABA-T and SSADH was also investigated. Moreover, hilicid-phospholipid complex was prepared, and its physic-chemical properties and oral pharmacokinetics in rats were investigated. The main results are as follows:(1) 4-Hydroxybenzaldehyde was shown to inhibit significantly GABA-T (IC50= 16.5μmol/L) in a competitive manner with respect toα-ketoglutarate but in a noncompetitive manner with respect to GABA. 4-Hydroxybenzaldehyde also exhibited a competitive inhibition of SSADH (IC50=24.7μmol/L). The inhibitory effects of 4-hydroxybenzaldehyde on both enzymes could result from the structural similarity between the molecule and the two enzymes'substrates, as well as the conjugative effect of benzene ring. Preliminary quantum chemical calculations suggested that the structural similarity of between 4-hydroxybenzaldehyde and the two substrates is due to the similarity of the energies and shapes of their molecular orbitals in nature. The results indicated that the presence of the benzene ring may be accepted by the active site of both enzymes, and 4-hydroxybenzaldehyde may be considered as a lead compound to design novel GABA-T and SSADH inhibitors.(2) Six groups of 4-hydroxybenzaldehyde analogues were examined as inhibitors for GABA-T and SSADH. Among them, syntheses of eight compounds were achieved in this laboratory. Investigation of 18 compounds including 4-hydroxybenzaldehyde revealed the significant structure-activity relations with regard to both enzymes inhibition. A carbonyl group or an aminomethyl group, as well as a hydroxy group at the para position of the benzene ring are important for GABA-T inhibition. A carbonyl group and a hydroxy group at the para position of the benzene ring are important for SSADH inhibition. IC50 values of 4-Acryloylphenol (6) for GABA-T and SSADH are 5.48 and 0.35μmol/L, respectively, and IC50 values of 4-β-chloropropionylphenol (7) for GABA-T and SSADH are 3.99 and 1.09μmol/L, exhibiting more potent activity than that of 4-hydroxybenzaldehyde. 4-Hydroxybenzylamine potently inhibited GABA-T (IC50= 15.4μmol/L) in a competitive manner with respect to GABA but in a noncompetitive manner with respect toα-ketoglutarate. A possible mechanism was speculated to rationalize the inhibition of GABA-T by 4-hydroxybenzaldehyde and 4-hydroxybenzylamine.(3) 4-Acryloylphenol was shown to inhibit irreversibly and potently the enzyme in a time-dependent manner with KI= 470μmol/L, kinact= 0.061 min-1 and kinact/KI= 0.129 (mmol/L)-1min-1. The inhibition was protected byα-ketoglutarate, indicating that it occurs at the active site of the enzyme.β-Mercaptoethanol also prevented the enzyme from irreversible inhibition. The possible mechanism involving a Michael addition was speculated to elucidate the inhibition. The rationality of the mechanism was supported by the preliminary quantum chemical calculations. The results suggested future directions for the design of more potent GABA-T inhibitors.(4) 4-β-Chloropropionylphenol was shown to inhibit irreversibly and potently the enzyme in a time-dependent manner with KI= 24.3μmol/L, kinact= 0.0467 min-1, kinact/KI= 1.88 (mmol/L)-1min-1. The inhibition was protected byα-ketoglutarate, indicating that it is active site-directed.β-Mercaptoethanol and glutathione also prevented the enzyme from irreversible inhibition. Since the published mechanism can't rationalize inactivation, a novel possible mechanism involving a nucleophilic substitution was speculated to rationalize the inactivation. Although the confirmation of the presumed mechanism requires further investigation, the results suggested that GABA-T could be inhibited irreversibly via a novel mechanism other than the two published mechanism, which give a new clue to design of more potent GABA-T inhibitors.(5) A group of flavonoids including baicalein, baicalin, scutellarein, scutellarin, quercetin, kaempferol and rutin, significantly and dose-dependently inhibited GABA-T and SSADH in a noncompetitive manner. The different structure-activity relations were observed with respect to inhibition of GABA-T and SSADH. Among the seven flavonoids, baicalein was the most potent inhibitor for GABA-T (IC50= 12.8μmol/L), and scutellarein exhibited the best inhibitory effect on SSADH (IC50= 7.2μmol/L). The results suggested that the inhibition of both enzymes by flavonoids may contribute to the beneficial effect of flavonoids on the central nervous system.(6) Helicid-phospholipid complex was prepare and the effect of such factors as the reactant concentrations, the ratio of reactants and the reactive time on the preparation was investigated via a orthogoral design, indicating the best conditions as follows: 5 mg/mL, 1: 5 and 2 h, respectively. X-ray diffraction spectra and DSC indicated that the complex exhibits an amorphous characteristic and change the transformation temperature of hilicid; 1H-NMR spectra and IR spectra suggested that hydroxy groups and aldehyde group of hilicid could interact with phospholipid. There is a great improvement in the solubility of hilicid-phospholipid complex both in water and in n-octanol. The complex significantly improved the relative bioavailability of helicid in rats. The improvement resulted from the particular physic-chemical and solubility properties of the hilicid-phospholipid complex.The results of this dissertation could be significantly useful to design novel GABA-T inhibitors, and will facilitate to elucidate the neuropharmacological actions of flavonoids. The research of helicid-phospholipid complex led to a valuable direction in development of novel formulations of natural drugs.