Probing the orthosteric binding site of GABAA receptors with heterocyclic GABA carboxylic acid bioisosteres.

The ionotropic GABAA receptors (GABAARs) are widely distributed in the central nervous system where they play essential roles in numerous physiological and pathological processes. A high degree of structural heterogeneity of the GABAAR has been revealed and extensive effort has been made to develop selective and potent GABAAR agonists. This review investigates the use of heterocyclic carboxylic acid bioisosteres within the GABAAR area. Several heterocycles including 3-hydroxyisoxazole, 3-hydroxyisoxazoline, 3-hydroxyisothiazole, and the 1- and 3-hydroxypyrazole rings have been employed in order to map the orthosteric binding site. The physicochemical properties of the heterocyclic moieties making them suitable for bioisosteric replacement of the carboxylic acid in the molecule of GABA are discussed. A variety of synthetic strategies for synthesis of the heterocyclic scaffolds are available. Likewise, methods for introduction of substituents into specific positions of the heterocyclic scaffolds facilitate the investigation of different regions in the orthosteric binding pocket in close vicinity of the core scaffolds of muscimol/GABA. The development of structural models, from pharmacophore models to receptor homology models, has provided more insight into the molecular basis for binding. Similar binding modes are proposed for the heterocyclic GABA analogues covered in this review by use of ligand-receptor docking into the receptor homology model and the presented structure-activity relationships. A network of interactions between the analogues and the binding pocket is leaving no room for substituents and underline the limited space in the GABAAR orthosteric binding site when in the agonist conformation.

SHOP: scaffold HOPping by GRID-based similarity searches.

A new GRID-based method for scaffold hopping (SHOP) is presented. In a fully automatic manner, scaffolds were identified in a database based on three types of 3D-descriptors. SHOP's ability to recover scaffolds was assessed and validated by searching a database spiked with fragments of known ligands of three different protein targets relevant for drug discovery using a rational approach based on statistical experimental design. Five out of eight and seven out of eight thrombin scaffolds and all seven HIV protease scaffolds were recovered within the top 10 and 31 out of 31 neuraminidase scaffolds were in the 31 top-ranked scaffolds. SHOP also identified new scaffolds with substantially different chemotypes from the queries. Docking analysis indicated that the new scaffolds would have similar binding modes to those of the respective query scaffolds observed in X-ray structures. The databases contained scaffolds from published combinatorial libraries to ensure that identified scaffolds could be feasibly synthesized.

Synthesis and pharmacological evaluation of 6-aminonicotinic acid analogues as novel GABA(A) receptor agonists.

A series of 6-aminonicotinic acid analogues have been synthesized and pharmacologically characterized at native and selected recombinant GABA(A) receptors. 6-Aminonicotinic acid (3) as well as 2- and 4-alkylated analogues (9-11, 14-16) display low to mid-micromolar GABA(A)R binding affinities to native GABA(A) receptors (K(i) 1.1-24 µM). The tetrahydropyridine analogue of 3 (22) shows low-nanomolar affinity (K(i) 0.044 µM) and equipotency as an agonist to GABA itself as well as the standard GABA(A) agonist isoguvacine. Cavities surrounding the core of the GABA binding pocket were predicted by molecular interaction field calculations and docking studies in a α1ß2γ2 GABA(A) receptor homology model, and were confirmed by affinities of substituted analogues of 3. The tight steric requirements observed for the remarkably few GABA(A)R agonists reported to date is challenged by our findings. New openings for agonist design are proposed which potentially could facilitate the exploration of different pharmacological profiles within the GABA(A)R area.

A unified model of the GABA(A) receptor comprising agonist and benzodiazepine binding sites.

We present a full-length α(1)ß(2)γ(2) GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α(1)R66, ß(2)T202, α(1)T129, ß(2)E155, ß(2)Y205 and the backbone of ß(2)S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α(1)R66, ß(2)T202, α(1)T129, ß(2)E155, ß(2)Y205 and ß(2)F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of ß(2)S156 and ß(2)Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α(1)T206 and γ(2)T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α(1)H101 and the N-methyl group near α(1)Y159, α(1)T206, and α(1)Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABA(A) receptor is made available as Model S1.

Synthesis and biological evaluation of 4-(aminomethyl)-1-hydroxypyrazole analogues of muscimol as γ-aminobutyric acid(a) receptor agonists.

A series of bioisosteric 4-(aminomethyl)-1-hydroxypyrazole (4-AHP) analogues of muscimol, a GABA(A) receptor agonist, has been synthesized and pharmacologically characterized at native and selected recombinant GABA(A) receptors. The unsubstituted 4-AHP analogue (2a) (EC(50) 19 µM, R(max) 69%) was a moderately potent agonist at human α(1)ß(2)γ(2) GABA(A) receptors, and in SAR studies substitutions in the 3- and/or 5-position were found to be detrimental to binding affinities. Ligand-receptor docking in an α(1)ß(2)γ(2) GABA(A) receptor homology model along with the obtained SAR indicate that 2a and muscimol share a common binding mode, which deviates from the binding mode of the structurally related antagonist series based on 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP, 1). Selectivity for α(1)ß(2)γ(2) over ρ(1) GABA(A) receptors was observed for the 5-chloro, 5-bromo, and 5-methyl substituted analogues of 2a illustrating that even small differences in structure can give rise to subtype selectivity.

SHOP: receptor-based scaffold HOPping by GRID-based similarity searches.

A new field-derived 3D method for receptor-based scaffold hopping, implemented in the software SHOP, is presented. Information from a protein-ligand complex is utilized to substitute a fragment of the ligand with another fragment from a database of synthetically accessible scaffolds. A GRID-based interaction profile of the receptor and geometrical descriptions of a ligand scaffold are used to obtain new scaffolds with different structural features and are able to replace the original scaffold in the protein-ligand complex. An enrichment study was successfully performed verifying the ability of SHOP to find known active CDK2 scaffolds in a database. Additionally, SHOP was used for suggesting new inhibitors of p38 MAP kinase. Four p38 complexes were used to perform six scaffold searches. Several new scaffolds were suggested, and the resulting compounds were successfully docked into the query proteins.