In silico screening of GABA aminotransferase inhibitors from the constituents of Valeriana officinalis by molecular docking and molecular dynamics simulation study.

Modulation of γ-aminobutyric acid (GABA) levels has been required in various disorders. GABA itself cannot be directly introduced into central nervous system (CNS) because of the blood brain barrier; inhibition of GABA aminotransferase (GABA-AT), which degrades GABA in CNS, has been the target for the modulation of GABA levels in CNS. Given that root extract of valerian (Valeriana officinalis) has been used for millennia as anti-anxiolytic and sedative, in silico approach was carried out to investigate valerian compounds exhibiting GABA-AT inhibiting activity. The 3D structure of human GABA-AT was created from pig crystal structure via homology modeling. Inhibition of GABA-AT by 18 valerian compounds was analyzed using molecular docking and molecular dynamics simulations and compared with known GABA-AT inhibitors such as vigabatrin and valproic acid. Isovaleric acid and didrovaltrate exhibited GABA-AT inhibiting activity in computational analysis, albeit less potent compared with vigabatrin. However, multiple compounds with low activity may have additive effects when the total extract of valeriana root was used in traditional usage. In addition, isovaleric acid shares similar backbone structure to GABA, suggesting that isovaleric acid might be a valuable starting structure for the development of more efficient GABA-AT inhibitors for disorders related with low level of GABA in the CNS.

Excavating precursors from the traditional Chinese herb Polygala tenuifolia and Gastrodia elata: Synthesis, anticonvulsant activity evaluation of 3,4,5-trimethoxycinnamic acid (TMCA) ester derivatives.

Epilepsy is a group of neurological disorders characterized by recurrent seizures that disturbs about 60 million people worldwide. In this article, a novel series of 3,4,5-trimethoxycinnamic acid (TMCA) ester derivatives 1-35 were designed inspired from the traditional Chinese herb pair drugs Polygala tenuifolia and Gastrodia elata and synthesized followed by in vivo and in silico evaluation of their anticonvulsant potential. All the synthesized derivatives were biologically evaluated for their anticonvulsant potential using two acute model of seizures induced in mice, the maximal electroshock (MES) and sc-pentylenetetrazole (PTZ) models. Simultaneously, the motor impairment as a surrogate of acute neurotoxicity and in vitro screening of cytotoxicity against HepG-2 cells line were assessed through the rotarod performance test and CCK-8 assay, respectively. In addition, the physicochemical and pharmacokinetic parameters of the active compounds were determined. Our results showed that compounds 5, 7, 8, 13, 20, 25, 28, 30 and 32 exhibited preferable anticonvulsant activity in primary evaluation, with compounds 28 and 32 being the most promising anticonvulsant agents in according to results of subsequent pharmacology and toxicity evaluation. Additionally, the molecular modeling experiments predicted good binding interactions of part of the obtained active molecules with the gamma-aminobutyric acid (GABA) transferas. Therefore, it could be concluded that the synthesized derivatives 28 and 32 would represent useful lead compounds for further investigation in the development of anticonvulsant agents.

Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase.

The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.

Inhibitory effect of Erigeron breviscapus extract and its flavonoid components on GABA shunt enzymes.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter, is metabolized by the successive action of GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Inhibition of both enzymes in brain tissues increases the GABA level and may have therapeutic applications in neurological diseases. Erigeron breviscapus ethanol extract was evaluated for their effect on both enzymes. This extract, its ethyl acetate fraction and aqueous fraction, significantly inhibited them at >100 microg/ml. Flavonoid components of E. breviscapus potently and noncompetitively inhibited both enzymes, and the different structure-activity relations were observed with respect to inhibition of both enzymes. Baicalein was the most potent inhibitor for GABA-T with an IC50 value of 12.8+/-1.2 microM, and scutellarein exhibited the best inhibitory effect on SSADH with an IC50 value of 7.20+/-0.9 microM. The present results may imply new pharmacological actions of E. breviscapus and contribute partially to the beneficial effect of the herb and flavonoids on the central nervous system.

Recombinant brain 4-aminobutyrate aminotransferases overexpression, purification, and identification of Lys-330 at the active site.

4-Aminobutyrate aminotransferase (4-aminobutyrate: 2-oxoglutarate aminotransferase EC 2.6.1.19) is a key enzyme of the 4-aminobutyric acid shunt. It catalyzes the conversion of 4-aminobutyrate to succinic semialdehyde. In an effort to clarify the structure-function relationships of 4-aminobutyrate aminotransferase, we analyzed 4-aminobutyrate aminotransferase cDNA from pig brain. The inclusion bodies were formed when recombinant 4-aminobutyrate aminotransferase was overexpressed in Escherichia coli. The unfolded overproduced proteins, were purified by hydroxylapatite chromatography in the presence of urea and refolded by a sequential dialysis method. The renatured protein regained its catalytic activity. The lysyl residue at the 330 position of the amino-acid sequence serves as the anchoring site of the cofactor pyridoxal 5'-P. To verify the catalytic site of 4-aminobutyrate aminotransferase, lysine 330 was mutated to arginine by site-specific mutagenesis. Overexpression and purification of the mutated 4-aminobutyrate aminotransferase (K330R) were performed by the same method used the purification of wild-type 4-aminobutyrate aminotransferase. The purified and renatured K330R protein did not show the catalytic activity of wild type 4-aminobutyrate aminotransferase. Furthermore, the mutated protein did not show any absorption band over the spectral range of 320-460 nm characteristic of pyridoxal 5'-P covalently linked to the protein. From the results presented here, it is concluded that lysine 330 is essential for the catalytic function of the aminotransferase.

A rat brain cDNA encodes enzymatically active GABA transaminase and provides a molecular probe for GABA-catabolizing cells.

cDNAs encoding gamma-aminobutyric acid aminotransferase (GABA-T) were isolated from a lambda ZAP rat hippocampal cDNA expression library by two independent cloning methods, immunological screening with an antimouse GABA-T antibody and plaque hybridization with a GABA-T cDNA probe derived by polymerase chain reaction. We have produced enzymatically active GABA-T from a rat brain cDNA containing the full-length GABA-T coding region. Our rat brain GABA-T cDNAs hybridize to mRNAs in brain and peripheral tissues, including liver, kidney, and testis. We have also detected GABA-T mRNA in GABAergic cells of rat cerebellar cortex by in situ hybridization. Our rat brain GABA-T probe hybridizes to Purkinje, basket, stellate, and Golgi II cells, the same GABAergic neurons previously shown to contain glutamate decarboxylase GAD65 and GAD67.