Design and mechanism of tetrahydrothiophene-based γ-aminobutyric acid aminotransferase inactivators

Abstract

Low levels of γ-aminobutyric acid (GABA), one of two major neurotransmitters that regulate brain neuronal activity, are associated with many neurological disorders, such as epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, and cocaine addiction. One of the main methods to raise the GABA level in human brain is to use small molecules that cross the blood-brain barrier and inhibit the activity of γ-aminobutyric acid aminotransferase (GABA-AT), the enzyme that degrades GABA. We have designed a series of conformationally restricted tetrahydrothiophene-based GABA analogues with a properly positioned leaving group that could facilitate a ring-opening mechanism, leading to inactivation of GABA-AT. One compound in the series is 8 times more efficient an inactivator of GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. Our mechanistic studies show that the compound inactivates GABA-AT by a new mechanism. The metabolite resulting from inactivation does not covalently bind to amino acid residues of GABA-AT but stays in the active site via H-bonding interactions with Arg-192, a π-π interaction with Phe-189, and a weak nonbonded S···O═C interaction with Glu-270, thereby inactivating the enzyme.

Figure 1

Metabolic cycle of L-glutamate

Figure 2

Vigabatrin analogs that follow one GABA-AT inactivation mechanism exclusively

Figure 3

Tetrahydrothiophene-based GABA analogs

Figure 4

The PLP-dihydrothiophene complex, shown in ball-and-stick form, is the final adduct, which contains a non-planar dihydrothiophene ring. Carbons are in beige, nitrogens are in blue, oxygens are in red, phosphorus is in orange, and sulfur is in yellow. (A) The electron density of the simulated annealing omit map (fo-fc) is shown as a grey mesh at 3.5 σ around the PLP-dihydrothiophone adduct. (B) and (C) are two different orientations of the same image: the electron density of the simulated omit map (fo-fc) is shown in green mesh at 4.1 σ around atoms in the dihydrothiophene ring (at a 0.6 Å radius). (B) This orientation displays the refined atom positions; (C) this orientation displays a buckled ring plane.

Figure 5

(A) Interactions between the PLP-dihydrothiophene adduct and nearby residues. (B) Directionality of the intermolecular weak nonbonded S···O interaction in theoretical studies, representing an n_O→ σ_s^* orbital interaction

Figure 6

Radioactive-labeling experiment for the inactivation of GABA-AT by 17: [7-³H]PLP-GABA-AT was prepared from apoGABA-AT and [7-³H]PLP then inactivated by 17, followed by denaturation and submission to HPLC. Fractions were collected each minute and counted for radioactivity. A solution of 1 mM PMP and 1 mM PLP was treated identically as a control.

Scheme 1

Mechanisms of inactivation of GABA-AT by vigabatrin

Scheme 2

Michael addition (pathway a) and enamine addition (pathway b) mechanisms for 17

Scheme 3

Syntheses of GABA Analogs 17 – 19

Scheme 4

Proposed mechanism for the inactivation of GABA-AT by 17

Scheme 5

Hydrolysis of Metabolite 34

Scheme 6

Synthesis of Cyclopentane Analog 39