Investigating the role of loop c hydrophilic residue 'T244' in the binding site of ρ1 GABAC receptors via site mutation and partial agonism

The loop C hydrophilic residue, threonine 244 lines the orthosteric binding site of ρ1 GABAC receptors was studied by point mutation into serine, alanine and cysteine, and tested with GABA, some representative partial agonists and antagonists. Thr244 has a hydroxyl group essential for GABA activity that is constrained by the threonine methyl group, orienting it toward the binding site. Significant decreases in activation effects of the studied ligands at ρ1 T244S mutant receptors, suggests a critical role for this residue. Results of aliphatic and heteroaromatic partial agonists demonstrate different pharmacological effects at ρ1 T244S mutant receptors when co-applied with GABA EC50 responses. ρ1 T244A and ρ1 T244C mutant receptors have minimal sensitivity to GABA at high mM concentrations, whereas, the ρ1 WT partial agonists, β-alanine and MTSEA demonstrate more efficacy and potency, respectively, than GABA at these mutant receptors. This study explores the role of Thr244 in the binding of agonists as an initial step during channel gating by moving loop C towards the ligand

Pharmacological Effect of GABA Analogues on GABA-<i>ϱ</i>2 Receptors and Their Subtype Selectivity

GABAϱ receptors are distinctive GABAergic receptors from other ionotropic GABAA and metabotropic GABAB receptors in their pharmacological, biochemical, and electrophysiological properties. Although GABA-ϱ1 receptors are the most studied in this subfamily, GABA-ϱ2 receptors are widely distributed in the brain and are considered a potential target for treating neurological disorders such as stroke. The structure of GABA-ϱ2 receptors and their pharmacological features are poorly studied. We generated the first homology model of GABA-ϱ2 channel, which predicts similar major interactions of GABA with the binding-site residues in GABA-ϱ1 and GABA-ϱ2 channels. We also investigated the pharmacological properties of several GABA analogues on the activity of GABA-ϱ2 receptors. In comparison to their pharmacological effect on GABA-ϱ1 receptors, the activation effect of these ligands and their potentiation/inhibition impact on GABA response have interestingly shown inter-selectivity between the two GABA-ϱ receptors. Our results suggest that several GABA analogues can be used as research tools to study the distinctive physiology of GABA-ϱ1 and GABA-ϱ2 receptors. Furthermore, their partial agonist effect may hold promise for the future discovery of selective modulatory agents on GABAA receptors

Target Based Designing of Anthracenone Derivatives as Tubulin Polymerization Inhibiting Agents: 3D QSAR and Docking Approach

Novel anthracenone derivatives were designed through in silico studies including 3D QSAR, pharmacophore mapping, and molecular docking approaches. Tubulin protein was explored for the residues imperative for activity by analyzing the binding pattern of colchicine and selected compounds of anthracenone derivatives in the active domain. The docking methodology applied in the study was first validated by comparative evaluation of the predicted and experimental inhibitory activity. Furthermore, the essential features responsible for the activity were established by carrying out pharmacophore mapping studies. 3D QSAR studies were carried out for a series of 1,5- and 1,8-disubstituted10-benzylidene-10H-anthracen-9-ones and 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-one derivatives for their antiproliferation activity. Based on the pattern recognition studies obtained from QSAR results, ten novel compounds were designed and docked in the active domain of tubulin protein. One of the novel designed compounds “N1” exhibited binding energy −9.69 kcal/mol and predicted Ki 78.32 nM which was found to be better than colchicine

Stoichiometric Post‐Modification of Hydrogel Microparticles Dictates Neural Stem Cell Fate in Microporous Annealed Particle Scaffolds

Microporous annealed particle (MAP) scaffolds are generated from assembled hydrogel microparticles (microgels). It has been previously demonstrated that MAP scaffold are porous, biocompatible, and recruit neural progenitor cells (NPCs) to the stroke cavity after injection into the stroke core. Here, the goal is to study NPC fate inside MAP scaffolds in vitro. To create plain microgels that can later be converted to contain different types of bioactivities, the inverse electron‐demand Diels–Alder reaction between tetrazine and norbornene is utilized, which allows the post‐modification of plain microgels stoichiometrically. As a result of adhesive peptide attachment, NPC spreading leads to contractile force generation which can be recorded by tracking microgel displacement. Alternatively, non‐adhesive peptide integration results in neurosphere formation that grows within the void space of MAP scaffolds. Although the formed neurospheres do not impose a contractile force on the scaffolds, they are seen to continuously transverse the scaffolds. It is concluded that MAP scaffolds  can be engineered to either promote neurogenesis or enhance stemness depending on the chosen post‐modifications of the microgels, which can be key in modulating their phenotypes in various applications in vivo.The inverse electron‐demand Diels–Alder reaction between tetrazine and norbornene is used to create plain microgels for stoichiometric post‐modification with various peptides to control neural progenitor cell growth and differentiation in vitro. YIGSR peptide presentation leads to neurosphere formation while IKVAV leads to stem cell spreading and differentiation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/174794/1/adma202201921_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/174794/2/adma202201921.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/174794/3/adma202201921-sup-0001-SuppMat.pd

Enhancement of the GABA EC₅₀ response by CACA at ρ1 WT and ρ1 T244S receptors.

<p>(A) Concentration response curve of the co-application of increasing concentrations of CACA in the presence of GABA EC₅₀ at ρ1 WT and ρ1 T244S receptors, (Data = Mean ± SEM, n = 5). Sample traces of CACA co-applied with GABA EC₅₀ at ρ1GABA_C (B) WT and (C) ρ1 T244S receptors.</p

Chemical structures of ligands used in this study.

<p>Chemical structures of ligands used in this study.</p

Effect of glycine, β-alanine and 5-aminovaleric acid on the GABA EC₅₀ response.

<p>Concentration response curves of GABA EC₅₀ in the presence of (A) glycine, (B) β-alanine and (C) 5-aminovaleric acid at ρ1 WT and ρ1 T244S receptors, (Data = Mean ± SEM, n = 5).</p

Investigating the Role of Loop C Hydrophilic Residue ‘T244’ in the Binding Site of ρ1 GABA_C Receptors via Site Mutation and Partial Agonism - Fig 14

<p><b>Docking studies of MSTEA and GABA</b> (A) in the WT ρ1 GABA_C homology model based on GluCl in open conformation. (B) Docking studies of GABA (white) and MTSEA (yellow) with ρ1 T244C homology model based on GluCl where thiol group of Cys244 is oriented away from the binding site (Chi1 = -64°).</p

Docking studies of isoguvacine in the orthosteric binding site of ρ1 GABA_C homology model.

<p>(A) H-bonds and (B) hydrophobic interactions predicted to be formed by isoguvacine and the ρ1 GABA_C receptor.</p

Activity of GABA, glycine, β-alanine and 5-aminovaleric acid at ρ1 WT and ρ1 T244S receptors.

<p>Concentration response curves of GABA, glycine, β-alanine and 5-aminovaleric acid at ρ1 WT (A) and ρ1 T244S (B) receptors, (Data = Mean ± SEM, n = 5).</p