Novel Allosteric Ligands of γ-Aminobutyric Acid Transporter 1 (GAT1) by MS Based Screening of Pseudostatic Hydrazone Libraries

Abstract

This study describes the screening of dynamic combinatorial libraries based on nipecotic acid as core structure with substituents attached to the 5- instead of the common 1-position for the search of novel inhibitors of the GABA transporter GAT1. The generated pseudostatic hydrazone libraries included a total of nearly 900 compounds and were screened for their binding affinities toward GAT1 in competitive mass spectrometry (MS) based Binding Assays. Characterization of the hydrazones with the highest affinities (with cis-configured rac-16gf bearing a 5-(1-naphthyl)­furan-2-yl residue and a four atom spacer being the most potent) in binding and uptake experiments revealed an allosteric interaction at GAT1, which was not reported for any other nipecotic acid derivative up to now. Therefore, the herein introduced 5-substituted nipecotic acid derivatives could serve as valuable tools for investigations of allosterically modulated GABA transport mediated by GAT1 and furthermore as starting point for a new class of GAT1 inhibitors.

MS‐Based Screening of 5‐Substituted Nipecotic Acid Derived Hydrazone Libraries as Ligands of the GABA Transporter 1

“…The hydrazine rac ‐ 15 was synthesized as shown in Scheme . To this end, ketone 17 was reacted with tert ‐butyl carbazate in the presence of sodium cyanoborohydride and acetic acid to yield the doubly tert ‐butyloxycarbonyl (Boc)‐protected hydrazine precursor rac ‐ 18 following a protocol for an analogous reaction from the literature . This reductive hydrazine formation reaction exclusively led to the synthesis of the desired cis ‐configured rac ‐ 18 (79 % yield); no trans ‐configured isomer could be recovered.…”

“…The individual hydrazone required for full‐scale competitive MS Binding Assays was synthesized as exemplified in Scheme in analogy to the previously described procedure . To this end, 1.0 equivalent of aldehyde ( 14 ) was combined with 1.1 equivalents of hydrazine ( rac ‐ 15 ) and 2.0 equivalents of sodium deuteroxide.…”

“…This experimental procedure was basically in accordance with a formerly published protocol for the generation and screening of pseudostatic combinatorial hydrazone libraries toward the membrane‐bound transport protein mGAT1 by means of MS Binding Assays (with certain adaptions as described in Ref. ).…”

“…Based on molecular modeling the unsubstituted ( R )‐nipecotic acid [( R )‐ 6 ] preferably adopts a binding pose in which the piperidine nitrogen atom faces the intracellular side, whereas for the larger inhibitor tiagabine ( 2 ) the binding pose of the piperidine ring is switched with the nitrogen and the attached arylalkyl residue facing the extracellular side of the transporter . On the basis of these results, we concluded that substitutions particularly at positions 4 or 5 with an appropriate spacer and lipophilic residue could yield GABA uptake inhibitors in which the binding pose of the piperidine ring is retained relative to that of unsubstituted ( R )‐ 6 . It is hoped that such compounds could bring new insight into the interactions between the ligand and target molecules or could even lead to novel GABA uptake inhibitors with higher potencies.…”

“…A conversion of this “C0” hydrazine, rac ‐ 15 , with appropriate aldehydes 14 would lead to hydrazones with a total spacer length of three atoms and thus complement the yet‐described series of hydrazones with a spacer between the 5‐position of the nipecotic acid and the lipophilic moiety comprising four (see for example, rac ‐ 13 ) and five atoms. Herein we describe the synthesis of rac ‐ 15 and its application as a building block for the generation of hydrazone libraries rac ‐ 16 (Scheme ), the latter of which were then screened toward the target mGAT1 in competitive MS Binding Assays . Potential hits were identified in deconvolution experiments of the most active libraries and verified by individually resynthesizing corresponding hydrazones followed by determination of their binding affinities (p K i values) in full‐scale competitive binding assays.…”