Novel Zn²⁺ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents

<div><p>Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion <i>in vivo</i>. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn²⁺ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn²⁺ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.</p></div

IPGTT in C57BL/6J mice; AZ7914 (<i>A</i>), AZ4237 (<i>B</i>), AZ1395 (<i>C</i>).

<p>Compounds (black triangles: 30 mg/kg, black circles: 100 mg/kg, grey circles: exendin-4, 1 μg/kg) or vehicle (white circles) were IP administered to fasted C57BL/6J mice at -30 min. Glucose solution (1.5 g/kg) was given IP at 0 min. Values are mean ± SEM (n = 7). ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 <i>versus</i> vehicle (repeated measures ANOVA followed by student’s t-test).*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>versus</i> vehicle (repeated measures ANOVA followed by Dunnett).</p

Plasma concentration–time profile of test compounds in the IPGTT using C57BL/6J mice; AZ7914 (<i>A</i>), AZ4237 (<i>B</i>), AZ1395 (<i>C</i>).

<p>Compounds were IP administered to fasted C57BL/6J mice at -30 min as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.g007" target="_blank">Fig 7</a> legend. Data represent the value of pooled samples from 7 animals.</p

Design of Selective sPLA₂‑X Inhibitor (−)-2-{2-[Carbamoyl-6-(trifluoromethoxy)‑1<i>H</i>‑indol-1-yl]pyridine-2-yl}propanoic Acid

A lead generation campaign identified indole-based sPLA₂-X inhibitors with a promising selectivity profile against other sPLA₂ isoforms. Further optimization of sPLA₂ selectivity and metabolic stability resulted in the design of (−)-<b>17</b>, a novel, potent, and selective sPLA₂-X inhibitor with an exquisite pharmacokinetic profile characterized by high absorption and low clearance, and low toxicological risk. Compound (−)-<b>17</b> was tested in an ApoE^–/– murine model of atherosclerosis to evaluate the effect of reversible, pharmacological sPLA₂-X inhibition on atherosclerosis development. Despite being well tolerated and achieving adequate systemic exposure of mechanistic relevance, (−)-<b>17</b> did not significantly affect circulating lipid and lipoprotein biomarkers and had no effect on coronary function or histological markers of atherosclerosis

Insulin secretion assays in mouse islets.

<p>Effect on insulin secretion by each compound. Islets were treated with 1 and 10 μM of AZ7914 (<i>A</i>), AZ4237 (<i>B</i>), AZ1395 (<i>C</i>). The assays were performed in the absence or presence of 5 μM of Zn²⁺. Values were calculated as the ratio of insulin concentration compared to the basal control and expressed as the average of three separate measurements ± SEM.</p

Binding parameters for Zn²⁺-interaction with AZ1395, AZ4237 and AZ7914 determined by ITC at 30°C.

<p>Binding parameters for Zn²⁺-interaction with AZ1395, AZ4237 and AZ7914 determined by ITC at 30°C.</p

Plasma concentration–time profile of test compounds in the IPGTT using DIO mice; AZ7914 (<i>A</i>), AZ4237 (<i>B</i>), AZ1395 (<i>C</i>).

<p>Compounds were IP administered to fasted DIO mice at -30 min as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.g008" target="_blank">Fig 8</a> legend. Data represent the value of pooled samples from 7 animals.</p

<i>In vivo</i> exposures <i>versus in vitro</i> potencies for AZ7914 (<i>A</i>), AZ4237 (<i>B</i>), AZ1395 (<i>C</i>).

<p>C_<i>u</i>,_<i>av</i> (μM) / EC₅₀ (μM) and C_<i>u</i>,_<i>av</i> (μM) / <i>K</i>_D (μM) calculated for different compound doses (30 or 100 mg/kg) IP administered to normal C57BL/6J mice. EC₅₀ and <i>K</i>_D values were from <i>in vitro</i> assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.t001" target="_blank">Table 1</a>) performed in the absence (w/o) or presence of 5 μM Zn²⁺ (+) and adjusted for protein binding. a) Compound weakly active in <i>in vitro</i> assay: Potency >33 μM, E_max ≥ 8%. b) Compound inactive in <i>in vitro</i> assay: Potency >33 μM, E_max < 2%. For bars labeled with a) and b), EC₅₀ = 33 μM has been used for calculations, resulting in overestimation of C_<i>u</i>,_<i>av</i> / EC₅₀ ratios. This is indicated with open bars. Closed bars indicate that EC₅₀ values have been determined to a defined value. ND, not determined.</p

Chemical structures of four GPR39 agonists.

<p>AZ7914, 6-(3-chloro-2-fluoro-benzoyl)-2-(2-methylthiazol-4-yl)-3,5,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-one; AZ1395, 3,4-bis(2-imidazol-1-ylethoxy)benzonitrile; AZ4237, 6-[(4-chlorophenyl)methyl]-7-hydroxy-5-methyl-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid.</p

AZ7914 (<i>A</i>), AZ4237 (<i>B</i>) and AZ1395 (<i>C</i>) ten point concentration response curves for <i>in vitro</i> screens.

<p>Concentration responses of compounds were measured with DMR, IP₁ and cAMP assays in the absence (-) or presence of 5 μM Zn²⁺ (+) as indicated in the figure. Lines represent fits to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.e001" target="_blank">Eq 1</a>. Cells employed were HEK293s-hGPR39 (hGPR39), untransfected HEK293s (HEK), and NIT-1 cells endogenously expressing GPR39. Responses were normalized to AZ1395 in each assay and presented as % effect of control. For HEK293s and HEK293s-hGPR39 cells, individual DMR assays were run in singlicates and IP₁ and cAMP assays were run in triplicates. Values shown are means ± SEM of typically three (two to five) independent experiments. For NIT-1 cells, IP₁ assays were typically run in duplicates (one to four experiments), and values are shown as means with error bars representing range. EC₅₀ values calculated from the data in Fig 2 are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145849#pone.0145849.t001" target="_blank">Table 1</a>.</p