Bromocryptine is a potent inhibitor that lacks an acidic side chain group like glutamate but contains homologous lipophilic residues along a peptide-like framework. In an effort to gain insight into the plausible interactions with VGLUT as a function of the QDC-based ligand design features (Fig. L-Glutamate is usually stored in synaptic vesicles prior to its depolarization-triggered, calcium-dependent release from neuron terminals1-4 and is transported into the vesicles in an ATP-dependent manner by the glutamate vesicular transporter (VGLUT). Unlike the plasma membrane neurotransmitter transporters, VGLUT is usually stimulated by low, physiologically relevant concentrations of Cl- ion,5 although the contribution of the Cl- – to pH has been debated.2-7 VGLUT is specific for glutamate but it is low affinity (Km = 1 to 3 mM), which contrasts with the plasma membrane transporters that are specific for glutamate but high affinity (Km = 5-50 M).8-11 To differentiate between these transporters, potent and selective inhibitors of VGLUT are needed. The main VGLUT inhibitor structures have been recently reviewed.1 In brief, aspartate5,12 and simple glutamate analogs are not good inhibitors of VGLUT, whereas some kynurenate analogs showed modest activity. The alkaloid bromocryptine (ki = 20 M) and certain azo dyes (e.g., trypan blue) are among the most potent VGLUT inhibitors (Fig. 1).13 We reported a systematic, structure-activity study of quinoline 2,4-dicarboxylic acids (QDCs; Fig . 2) as inhibitors14, 15 that seeded the development of the first pharmacophore model1 for VGLUT and the use of QDCs as a key motif for future inhibitor design and substituent variation. Open in a separate window Fig 1 Structures of VGLUT inhibitors Open in a separate window Fig 2 Proposed QDC inhibitor structural relationship to peptides. Some of the more potent QDC-based inhibitors contained lipophilic groups at position 6 or a hydroxyl at position 8. Combining these favorable substituents into the QDC template led to the observation that this pattern overlays with a peptide that contains (HO2C)WEX(NH2) (Fig. 2). The very weak basicity of the QDC nitrogen also suggested that a peptide amide might be an appropriate isostere. This prompted an investigation of small peptides that might be capable of binding VGLUT. Peptide-based inhibitors are also possible leads to uncover protein interactions. Based on observations that QDCs made up of an embedded glutamate moiety and lipophilic substituents (phenyl, styryl, Proglumide etc.) confer greater inhibitory activity, a tetrapeptide library was envisioned in which the C-terminus amino acid was occupied by either tryptophan (W) or phenylalanine (F) to represent the lipophilic substituent and the adjacent position occupied by a glutamate (E) residue. The N-terminus and second residue (X1 and X2) were systematically varied to investigate how these positions could enhance binding (Fig. 3). Open in a separate window Fig. 3 Tetrapeptide design based on the QDC-template. To further refine our binding requirements and increase the overall library diversity, either d- or l-amino acids were used. Further rationale for the incorporation of d-glutamate into the libraries is based on the modest activity of this enantiomer as an inhibitor of VGLUT.11 Overall, stereoisomeric tetrapeptides X1X2EW(F) were prepared and evaluated as VGLUT inhibitors (Fig. 3; Table 1). Table 1 Inhibition of VGLUT by Tetrapeptides1 thead th align=”center” rowspan=”1″ colspan=”1″ X1 /th th align=”center” rowspan=”1″ colspan=”1″ X2 /th th align=”center” rowspan=”1″ colspan=”1″ X3 /th th align=”center” rowspan=”1″ colspan=”1″ X4 /th th align=”center” rowspan=”1″ colspan=”1″ 3H-lGlu uptake br / (% of control)2 /th /thead em Library 1 /em AA3AAEWQAAEW56 1%QWEW66 4%QIEW38 5%D-QD-IL -ED-W35 3%L-QD-IL-ED-W28 3% em Library 2 /em AAAAEFNAAEF63 17%WAAEF36 2%WNEF13 3%D-WL-ND-ED-F41 1% em Other /em Congo Red (2 M)31 2% Open in a separate window 1 Tetrapeptides tested as racemic mixtures at 2mM. 2 Control rate for 3H-L-glutamate uptake was 1847130(n=17) pmol/min/mg protein. 3 AA = 19 different amino acids. em Peptide Synthesis /em . 16 Tetrapeptides were synthesized according to Scheme 1 and structures determined by NMR and/or mass spectrometry. Open in a separate window Scheme 1 Synthesis of target tetrapeptides made up of a glutamate at position 3. em Inhibition of VGLUT by Tetrapeptides /em . 17 Screening of the peptide libraries as VGLUT inhibitors was carried out using 3H-glutamate as substrate and the ability of test compounds to block the uptake of 3H-glutamate into synaptic vesicles isolated from rat forebrain. Tetrapeptide sub-libraries of the type X1X2EW (Library 1) or X1X2EF (Library 2), where X1 and X2 were varied as amino acids (AA) in D- or L- form (except cysteine), and where the identity of X1 was Proglumide known, were tested as inhibitors of VGLUT.18 The sub-libraries were screened and the pools showing the most inhibition of uptake were.Since small peptides may assume a host of conformations, a plausible extended conformation of lQdIlEdW was selected for a VGLUT pharmacophore model comparison. Analysis of the lQdIlEdW conformation alignment reveals that this N-terminal arginine (Q) correlates to the -carboxyl model region, the glutamic acid (E) side chain carboxyl moiety corresponds to the pharmacophore H-bonding acceptor group, and the C-terminal tryptophan (W) is consistent with the aromatic ring lipophilic pocket superposition area. calcium-dependent release from neuron terminals1-4 and is transported into the vesicles in an ATP-dependent manner by the glutamate vesicular transporter (VGLUT). Unlike the plasma membrane neurotransmitter transporters, VGLUT Proglumide is stimulated by low, physiologically relevant concentrations of Cl- ion,5 although the contribution of the Cl- – to pH has been debated.2-7 VGLUT is specific for glutamate but it is low affinity (Km = 1 to 3 mM), which contrasts with the plasma membrane transporters that are specific for glutamate but high affinity (Km = 5-50 M).8-11 To differentiate between these transporters, potent and selective inhibitors of VGLUT are needed. The main VGLUT inhibitor structures have been recently reviewed.1 In brief, aspartate5,12 and simple glutamate analogs are not good inhibitors of VGLUT, whereas some kynurenate analogs showed modest activity. The alkaloid bromocryptine (ki = 20 M) and certain azo dyes (e.g., trypan blue) are among the most potent VGLUT inhibitors (Fig. 1).13 We reported a systematic, structure-activity study of quinoline 2,4-dicarboxylic acids (QDCs; Fig . 2) as inhibitors14, 15 that seeded the development of the first pharmacophore model1 for VGLUT and the use of QDCs as a key motif for future inhibitor design and substituent variation. Open in a separate window Fig 1 Structures of VGLUT inhibitors Open in a separate window Fig 2 Proposed QDC inhibitor structural relationship to peptides. Some of the more potent QDC-based inhibitors contained lipophilic groups at position 6 or a hydroxyl at position 8. Combining these favorable substituents into the QDC template led to the observation that this pattern overlays with a peptide that contains (HO2C)WEX(NH2) (Fig. 2). The very weak basicity of the QDC nitrogen also suggested that a peptide amide might be an appropriate isostere. This prompted an investigation of small peptides that might be capable of binding VGLUT. Peptide-based inhibitors are also possible leads to uncover protein interactions. Based on observations that QDCs containing an embedded glutamate moiety and lipophilic substituents (phenyl, styryl, etc.) confer greater inhibitory activity, a tetrapeptide library was envisioned in which the C-terminus amino acid was occupied by either tryptophan (W) or phenylalanine (F) to represent the lipophilic substituent and the adjacent position occupied by a glutamate (E) residue. The N-terminus and second residue (X1 and X2) were systematically varied to investigate how these positions could enhance binding (Fig. 3). Open in a separate window Fig. 3 Tetrapeptide design based on the QDC-template. To further refine our binding requirements and increase the overall library diversity, either d- or l-amino acids were used. Further rationale for the incorporation of d-glutamate into the libraries is based on the modest activity of this enantiomer as an inhibitor of VGLUT.11 Overall, stereoisomeric tetrapeptides X1X2EW(F) were prepared and evaluated as VGLUT inhibitors (Fig. 3; Table 1). Table 1 Inhibition of VGLUT by Tetrapeptides1 thead th align=”center” rowspan=”1″ colspan=”1″ X1 /th th align=”center” rowspan=”1″ colspan=”1″ X2 /th th align=”center” rowspan=”1″ colspan=”1″ X3 /th th align=”center” rowspan=”1″ colspan=”1″ X4 /th th align=”center” rowspan=”1″ colspan=”1″ 3H-lGlu uptake br / (% of control)2 /th /thead em Library 1 /em AA3AAEWQAAEW56 1%QWEW66 4%QIEW38 5%D-QD-IL -ED-W35 3%L-QD-IL-ED-W28 3% em Library 2 /em AAAAEFNAAEF63 17%WAAEF36 2%WNEF13 3%D-WL-ND-ED-F41 1% em Other /em Congo Red (2 M)31 2% Open in a separate window 1 Tetrapeptides tested as racemic mixtures at 2mM. 2 Control rate for 3H-L-glutamate uptake was 1847130(n=17) pmol/min/mg protein. 3 AA = 19 different amino acids. em Peptide Synthesis /em . 16 Tetrapeptides were synthesized according to Scheme 1 and structures determined by NMR and/or mass spectrometry. Open in a separate window Scheme 1 Synthesis of target tetrapeptides containing a glutamate at position 3. em Inhibition of VGLUT by Tetrapeptides /em . 17 Screening of the peptide libraries as VGLUT inhibitors was carried out using 3H-glutamate as substrate and the ability of test compounds to block the uptake of 3H-glutamate into Rabbit polyclonal to ATF2 synaptic vesicles isolated from rat forebrain. Tetrapeptide sub-libraries of the type X1X2EW (Library 1) or X1X2EF (Library 2), where X1.