Structure-Based Discovery of 1<i>H</i>‑Indazole-3-carboxamides as a Novel Structural Class of Human GSK‑3 Inhibitors

An in silico screening procedure was performed to select new inhibitors of glycogen synthase kinase 3β (GSK-3β), a serine/threonine protein kinase that in the last two decades has emerged as a key target in drug discovery, having been implicated in multiple cellular processes and linked with the pathogenesis of several diseases. GSK-3β inhibitors might prove useful as therapeutic compounds in the treatment of conditions associated with elevated levels of enzyme activity, such as type-2 diabetes and neurological disorders, for example, Alzheimer’s disease, bipolar disorder, neuronal cell death, stroke, and depression. In this work, virtual screening studies were applied to proprietary compound libraries, and the functional activities of selected compounds were assayed on human GSK-3β. The in silico screening procedure enabled the identification of eight hit compounds showing pIC₅₀ values ranging from 4.9 to 5.5. X-ray crystallographic studies resulted in a 2.50 Å three-dimensional structure of GSK-3β complexed with one of the selected compounds, confirming that the inhibitor interacts with the enzyme according to the docking hypothesis. Importantly, molecular docking was able to find a new chemical scaffold for GSK-3β inhibition, providing grounds for rational structure-based design aimed at further optimization of the initial hits

The mPGES-1 inhibitor AF3485 reduces A431 tumor growth in xenograft nude mice.

<p>(A) Tumor volume measured in athymic mice inoculated with A431 cells and treated with vehicle (Ctr, 0.5% MTC), AF3485 (20, 1, or 0.1 mg/kg/mouse), or AG1478 (400 µg/mouse). (B) EGFR phosphorylation in xenograft tumor tissues reported as optical density (OD  =  ratio between phospho-tyr and EGFR expression). *P<0.05 vs Cont. (C) Representative images of the proliferative Ki67 index in tumor sections.</p

The mPGES-1 inhibitor AF3485 reduces VEGF and FGF-2 expression in tumors and in A431 cells.

<p>(A) VEGF and FGF-2 levels in tumors specimens and (B) in A431 cells treated with or without IL-1β (10 ng/ml, 18 h) in presence/absence of AF3485 (10 µM). *P<0.05, **P<0.01. ADU =  arbitrary density unit ± standard deviation. (C) Pseudocapillary formation of EC exposed for 16 h to conditioned media from A431 treated with 0.1% FBS (panel a: control), IL-1β (panel b: 10 ng/ml, 18 h), AF3485 (panel c: 10 µM, 24 h), IL-1β + AF3485 (panel d: 24 h) *P<0.01 vs. cont, #p<0.01 vs. IL-1β. (D) VEGF and FGF-2 expression in A431 cells co-cultured with NIH-3T3 for 24 h.</p

AF3485 inhibits tumor growth and angiogenesis.

<p>Representative images of histological analysis of CD31 in tumor sections from (a) control, (b) AF3485-treated mice. Images taken at 40X. Quantification of microvessel density in tumors. **P<0.01 vs Cont.</p

Effects of AF3485 on PGE2 release and growth in non tumor cells.

<p>Human fibroblasts (HF), human endothelial cells (EC) and mouse fibroblasts (NIH-3T3) were exposed to AF3485 (1–10 µM), and PGE₂ release and cell growth was evaluated by EIA and MTT assay, respectively.</p

Effects of AF3485 on leukotriene biosynthesis.

<p>A23187 (5 µM) stimulated-HL-60 cells were exposed to AF3485 (10 µM) for 30 min at 37°C, then LTB4 and LTC4 secretion was measured by EIA. Nordihydroguiaretic acid (NDGA) and (Phorbol 12-myristate 13-acetate) PMA were used as reference compounds. Inhibition values were obtained in 3 separate experiments.</p

AF3485 inhibits IL-1β-induced PGE₂ production in A431 cells.

<p>(A) COX-1, -2 and mPGES-1 mRNA expression in A431 cells in presence/absence of IL-1β (10 ng/ml, 18 h). Data reported as fold increase vs. unstimulated control. **P<0.01 vs Control. (B) Western blot of COX-1, -2, and mPGES-1 expression in A431 cells in presence/absence of IL-1β (10 ng/ml, 18 h). (C) PGE₂ levels in unstimulated A431 cells treated with AF3485 (0–100 µM, 30 min). PGE₂ levels measured by ELISA (24 h from plating.) Data reported as ng/ml. *P<0.05; **P<0.01; ***P<0.001 vs Control. (D) PGE₂ levels in unstimulated or IL-1β (10 ng/ml, 24 h) pre-treated A431 cells, and treated with AF3485 (10 µM, 30 min). The data are reported as ng/ml. **P<00.1 vs Cont; ##P<0.01 vs Cont; §§P<0.01 vs IL-1β (10 ng/ml).</p

AF3485 inhibits human recombinant mPGES-1 activity in vitro and selectively inhibits IL-1β-induced PGE₂ production in A549 cells.

<p>Structure of AF3485 (A). Effect of AF3485 (B) on hmPGES-1 expressed in bacterial membrane. A549 cells pre-treated with IL-1β (10 ng/ml, 18 h) then treated with AF3485 (C) (0.01–100 µM, 30 min). PGE₂ and PGF_2α levels measured by ELISA. Data reported as % inhibition of PGE₂ (□) or PGF_2α levels (▿).</p

Hit Optimization of 5‑Substituted‑<i>N</i>‑(piperidin-4-ylmethyl)‑1<i>H</i>‑indazole-3-carboxamides: Potent Glycogen Synthase Kinase‑3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders

Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3β (GSK-3β) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3β inhibitors having the common <i>N</i>-[(1-alkylpiperidin-4-yl)­methyl]-1<i>H</i>-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound <b>5</b> with GSK-3β. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3β inhibitors with good cell activity. Among the compounds assessed in the <i>in vivo</i> PK experiments, <b>14i</b> showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound <b>14i</b> was selected for further <i>in vitro</i>/<i>in vivo</i> pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3β inhibitors as new tools in the development of new treatments for mood disorders

Discovery and Pharmacological Profile of New 1<i>H</i>‑Indazole-3-carboxamide and 2<i>H</i>‑Pyrrolo[3,4‑<i>c</i>]quinoline Derivatives as Selective Serotonin 4 Receptor Ligands

Since the discovery of the serotonin 4 receptor (5-HT₄R), a large number of receptor ligands have been studied. The safety concerns and the lack of market success of these ligands have mainly been attributed to their lack of selectivity. In this study we describe the discovery of <i>N</i>-[(4-piperidinyl)­methyl]-1<i>H</i>-indazole-3-carboxamide and 4-[(4-piperidinyl)­methoxy]-2<i>H</i>-pyrrolo­[3,4-<i>c</i>]­quinoline derivatives as new 5-HT₄R ligands endowed with high selectivity over the serotonin 2A receptor and human ether-a-go-go-related gene potassium ion channel. Within these series, two molecules (<b>11ab</b> and <b>12g</b>) were identified as potent and selective 5-HT₄R antagonists with good in vitro pharmacokinetic properties. These compounds were evaluated for their antinociceptive action in two analgesia animal models. <b>12g</b> showed a significant antinociceptive effect in both models and is proposed as an interesting lead compound as a 5-HT₄R antagonist with analgesic action