Analysis of Differential Efficacy and Affinity of GABA_A (α1/α2) Selective Modulators

Selective modulators of the γ-amino butyric acid (GABA_A) family of receptors have the potential to treat a range of disease states related to cognition, pain, and anxiety. While the development of various α subunit-selective modulators is currently underway for the treatment of anxiety disorders, a mechanistic understanding of the correlation between their bioactivity and efficacy, based on ligand–target interactions, is currently still lacking. In order to alleviate this situation, in the current study we have analyzed, using ligand- and structure-based methods, a data set of 5440 GABA_A modulators. The Spearman correlation (ρ) between binding activity and efficacy of compounds was calculated to be 0.008 and 0.31 against the α1 and α2 subunits of GABA receptor, respectively; in other words, the compounds had little diversity in structure and bioactivity, but they differed significantly in efficacy. Two compounds were selected as a case study for detailed interaction analysis due to the small difference in their structures and affinities (Δp<i>K</i>_i_{(comp1_α1 – comp2_α1)} = 0.45 log units, Δp<i>K</i>_i_{(comp1_α2 – comp2_α2)} = 0 log units) as compared to larger relative efficacies (ΔRE_{(comp1_α1 – comp2_α1)} = 1.03, ΔRE_{(comp1_α2 – comp2_α2)} = 0.21). Docking analysis suggested that His-101 is involved in a characteristic interaction of the α1 receptor with both compounds <b>1</b> and <b>2</b>. Residues such as Phe-77, Thr-142, Asn-60, and Arg-144 of the γ chain of the α1γ2 complex also showed interactions with heterocyclic rings of both compounds <b>1</b> and <b>2</b>, but these interactions were disturbed in the case of α2γ2 complex docking results. Binding pocket stability analysis based on molecular dynamics identified three substitutions in the loop C region of the α2 subunit, namely, G200E, I201T, and V202I, causing a reduction in the flexibility of α2 compared to α1. These amino acids in α2, as compared to α1, were also observed to decrease the vibrational and dihedral entropy and to increase the hydrogen bond content in α2 in the apo state. However, freezing of both α1 and α2 was observed in the ligand-bound state, with an increased number of internal hydrogen bonds and increased entropy. Therefore, we hypothesize that the amino acid differences in the loop C region of α2 are responsible for conformational changes in the protein structure compared to α1, as well as for the binding modes of compounds and hence their functional signaling

Kinase selectivity screening.

<p>Compounds were screened in one of two kinase panels at a concentration of 10 µM. The degree of inhibition of each kinase is indicated by shading as follows: white <29% inhibition, light grey 30-49% inhibition, dark grey 50-69% inhibition, black >70% inhibition. Key to compounds: (1) 0181276, (2) CE-160042, (3) 0180532, (4) PF-4903528, (5) PF-4936572, (6) PF-4995633, (7) AG-24290, (8) PF-4279731, (9) CE-317112, (10) PF-1242377, (11) PF-744923, (12) PF-2368935, (13) PF-956933. NT  =  not tested.</p

Modelling of the binding-site residues with putative inhibitors.

<p>Compounds (orange) docked into the catalytic domain of the crystal structure of <i>Hs</i>GSK3 beta in their binding modes. A: CE-317112 shows preference for <i>Hs</i>GSK-3 beta. B: PF-4903528 shows preference for <i>Tbru</i>GSK-3 short. The residues that differ between human and <i>Tbru</i>GSK-3 short are shown in magenta, with only L132M (top centre of the image) directly lining the pocket. Images were created using the Pfizer molecule-modelling package MoViT.</p

GSK-3 Enzyme, Antiparasitic and cytotoxicity testing (values in µM).

<p>Values are a mean of at least 2 replicates. NT  =  not tested due to limited compound availability.</p><p>*Due to limited compound availability, these compounds were only tested at a single concentration of 1 µM and showed >50% inhibition at this concentration.</p

Discovery of a Series of Indazole TRPA1 Antagonists

A series of TRPA1 antagonists is described which has as its core structure an indazole moiety. The physical properties and <i>in vitro</i> DMPK profiles are discussed. Good <i>in vivo</i> exposure was obtained with several analogs, allowing efficacy to be assessed in rodent models of inflammatory pain. Two compounds showed significant activity in these models when administered either systemically or topically. Protein chimeras were constructed to indicate compounds from the series bound in the S5 region of the channel, and a computational docking model was used to propose a binding mode for example compounds

Anti-nicotine antibody titer and function in mice.

<p>Panel A: BALB/c mice (n = 12/gp) were immunized by IM injection with 10 µg of different nicotine Hapten-DT conjugates adjuvanted with Al(OH)₃ (40 µg Al³⁺) + CpG 24555 (50 µg) on days 0, 28 and 42. Plasma was collected on day 54 and anti-nicotine antibody levels determined by ELISA (Panel A). On day 56 animals received an IV injection of ³H-nicotine (0.05 mg/kg) and plasma and brains collected. Panel B shows nicotine levels in plasma (ng-eq/mL), and Panel C shows nicotine levels in brain (ng-eq/g).</p

General scheme for addition of different linkers to Hapten 7.

<p>General scheme for addition of different linkers to Hapten 7.</p

Structures of different linker-modified nicotine hapten.

<p>Structures of different linker-modified nicotine hapten.</p

Synthesis of Hapten 8.

<p>Synthesis of Hapten 8.</p

Synthesis of Hapten 5.

<p>Synthesis of Hapten 5.</p