A new VGLUT-specific potent inhibitor: pharmacophore of Brilliant Yellow

Abstract

The increased concentration of glutamate in synaptic vesicles, mediated by the vesicular glutamate transporter (VGLUT), is an initial vital step in glutamate synaptic transmission. Evidence indicates that aberrant overexpression of VGLUT is involved in certain pathophysiologies of the central nervous system. VGLUT is subject to inhibition by various types of agents. The most potent VGLUT-specific inhibitor currently known is Trypan Blue, which is highly charged, hence membrane-impermeable. We have sought a potent, VGLUT-specific agent amenable to easy modification to a membrane-permeable analog. We provide evidence that Brilliant Yellow exhibits potent, VGLUT-specific inhibition, with a Ki value of 12 nM. Based upon structure-activity relationship studies and molecular modeling, we have defined the potent inhibitory pharmacophore of Brilliant Yellow. This study provides new insight into development of a membrane-permeable agent to lead to specific blockade, with high potency, of accumulation of glutamate into synaptic vesicles in neurons.

Fig.1

Brilliant Yellow and Direct Violet 51 are potent inhibitors of vesicular glutamate uptake. Rat brain synaptic vesicles were pre-incubated in the presence of various concentrations of Brilliant Yellow (left) or Direct Violet 51 (right) in a mixture described in Materials and Methods, followed by addition of a mixture of [³H]glutamate ± ATP, and an additional 10-min incubation.

Fig. 2

Brilliant Yellow inhibits vesicular glutamate uptake without affecting GABA uptake. Bovine brain synaptic vesicles were pre-incubated in the presence of various concentrations of Brilliant Yellow, and further incubated with 2 mM ATP and 50 μM [³H]glutamate (left) or [³H]GABA (right), as described in Materials and Methods.

Fig. 3

Brilliant Yellow is a competitive inhibitor with glutamate for VGLUT. Synaptic vesicles were pre-incubated with 0, 40, and 80 nM Brilliant Yellow, followed by addition of a mixture of ATP and various concentrations of [³H]glutamate, with an additional 1.5- min incubation, as described in Materials and Methods. This represents a mean of three experimental data. v, initial rate of vesicular glutamate uptake; BY, Brilliant Yellow.

Fig. 4

Dose-response curve of 4,4’-dibenzamidostilbene-2,2’-disulfonate for vesicular glutamate uptake. Rat brain synaptic vesicles were incubated with [³H]glutamate ± ATP in the presence of various concentrations of 4,4’-dibenzamidostilbene-2,2’-disulfonate, as described in Fig. 1.

Fig. 5

IR-1048 reverses inhibition by Brilliant Yellow. Synaptic vesicles were incubated with [³H]glutamate, ATP, and various concentrations of IR-1048 in the absence (open circle) or presence (closed circle) of 0.3 μM Brilliant Yellow, as described in Fig. 1.

Fig. 6

IR-1048 does not reverse inhibition by IPF. Synaptic vesicles were incubated with [³H]glutamate, ATP, and various concentration of IR-1048 in the absence or presence of partially purified IPF (hydroxyapatite chromatography fraction 0.24 mg/ml), as described in Fig. 1.

Fig. 7

Specific action of Brilliant Yellow. Brilliant Yellow (BY) specifically inhibits VLGUT with high potency. Glu, glutamate; SV, synaptic vesicle.