A Novel Diphenylthiosemicarbazide Is a Potential Insulin Secretagogue for Anti-Diabetic Agent

<div><p>Insulin secretagogues are used for treatment of type 2 diabetes. We attempted to discover novel small molecules to stimulate insulin secretion by using in silico similarity search using sulfonylureas as query, followed by measurement of insulin secretion. Among 38 compounds selected by in silico similarity search, we found three diphenylsemicarbazides and one quinolone that stimulate insulin secretion. We focused on compound 8 (C8), which had the strongest insulin-secreting effect. Based on the structure-activity relationship of C8-derivatives, we identified diphenylthiosemicarbazide (DSC) 108 as the most potent secretagogue. DSC108 increased the intracellular Ca²⁺ level in MIN6-K8 cells. Competitive inhibition experiment and electrophysiological analysis revealed sulfonylurea receptor 1 (SUR1) to be the target of DSC108 and that this diphenylthiosemicarbazide directly inhibits ATP-sensitive K⁺ (K_ATP) channels. Pharmacokinetic analysis showed that DSC108 has a short half-life in vivo. Oral administration of DSC108 significantly suppressed the rises in blood glucose levels after glucose load in wild-type mice and improved glucose tolerance in the Goto-Kakizaki (GK) rat, a model of type 2 diabetes with impaired insulin secretion. Our data indicate that DSC108 is a novel insulin secretagogue, and is a lead compound for development of a new anti-diabetic agent.</p></div

Insulin secretory properties of DSC108.

<p>(A) Effect of C8 and DSC108 on insulin secretion from MIN6-K8 cells. Cells were stimulated by each concentration of compound in the presence of 11.2 mM glucose for 30 min. Data are fold-increase in insulin secretion relative to vehicle. Values are expressed as mean ± SEM (n = 3 for each compound). *P < 0.05, **P < 0.01 (Student unpaired <i>t</i> test). (B) Effect of DSC108 at 10 μM on the dynamics of insulin secretion from mouse perfused pancreata in the presence of 2.8 mM glucose. Values are expressed as mean ± SEM (n = 3).</p

Chemical structures of DSC108 and its sodium-salt form (DSC108-Na).

<p>Chemical structures of DSC108 and its sodium-salt form (DSC108-Na).</p

Effects of DSC108 and DSC108-Na on β-cell K_ATP channels.

<p><b>(</b>A) Inhibition of [³H]glibenclamide binding to human SUR1 by DSC108-Na. [³H]glibenclamide binding to human SUR1 is displaced by unlabeled DSC108-Na. Values are presented as mean ± SEM (n = 4). (B) Actual current traces recorded from COS-1 cells transfected with human SUR1 and human Kir6.2. The quasi-steady-state membrane current was recorded in the voltage-clamp mode using the ramp-pulse protocol (Inset), and plotted against membrane potential. Both DSC108 (3 μM) and glibenclamide (0.003 μM) inhibited the diazoxide (300 μM)-induced outward current. (C) Concentration-response curves for the inhibitory effects of DSC108, gliclazide, and glibenclamide on diazoxide (300 μM)-induced potassium current at 0 mV. The IC₅₀ values for the inhibitory effects of DSC108, gliclazide, and glibenclamide on the diazoxide-induced current are 2.3 μM, 0.86 μM and 0.003 μM, respectively. Each point represents mean ± SEM of 1–6 cells.</p

Insulin secretagogues identified by in silico screening in combination with insulin secretion measurement.

<p>(A) Insulin secretion from MIN6-K8 cells stimulated by 10 μM of each compound in the presence of 11.2 mM glucose. Data are fold-increase in insulin secretion relative to vehicle. Values are expressed as mean ± SEM (n = 3 for each compound). (B) Chemical structures of hit compounds. C8, 9, and 22 are diphenylsemicarbazides; C25 is a quinolone.</p

Effects of DSC108 and glibenclamide on the membrane potential of pancreatic β-cells.

<p>Membrane potential recording of isolated β-cells was performed by the patch-clamp method in the current clamp mode. (A) Representative membrane potential changes after treatment with 30 μM DSC108 or 1 μM glibenclamide. (B) Summarized data of membrane potential changes after treatment with 30 μM DSC108 (n = 7) or 1 μM glibenclamide (n = 4). Values are expressed as mean ± SEM. **P < 0.01 vs. pretreatment (Student paired <i>t</i> test).</p

Effects of DSC108-Na in vivo.

<p>(A) Plasma concentrations of DSC108-Na and gliclazide. 30 mg/kg of each compound was orally administered to wild type mice (n = 3 for each group) and blood samples were collected every 30 min for 2 hours from the tail vein. Concentration of the compound was analyzed by LC-MS. (B) Insulinotropic effect of DSC108-Na in vivo. 30 mg/kg of DSC108-Na was orally administered to wild type mice and plasma insulin concentrations were measured. Data are expressed as mean ± SEM (n = 4 for each group). **P < 0.01 (Student unpaired <i>t</i> test). (C) Glucose-lowering effect of DSC108-Na in OGTT. Changes in blood glucose levels after oral glucose load following administration of DSC108-Na (left). Vehicle or each concentration of DSC108-Na was administered orally at -20 min and glucose (1.5g/kg) was administered orally at 0 min. AUC of glucose is represented in bar graphs (right). Data are expressed as mean ± SEM (n = 4 for each group). Arrow and arrowhead indicate the administration of compound and glucose, respectively. *P < 0.05, **P < 0.01 vs. vehicle group (Dunnet’s method).</p

Improvement of glucose tolerance in GK rats by DSC108-Na.

<p>(A) Insulinotropic effect of DSC108-Na in vivo. 30 mg/kg of DSC108-Na was orally administered to GK rats and plasma insulin concentrations were measured. Data are expressed as mean ± SEM (n = 6 for each group). *P < 0.05 (paired <i>t</i> test). (B) Changes in blood glucose levels after oral glucose load following administration of DSC108-Na in GK rats (left). Vehicle or 100 mg/kg of DSC108-Na was orally administered to rats at -20 min, and glucose was orally loaded at 0 min. AUC of glucose is represented in bar graphs (right). Data are expressed as mean ± SEM (n = 6 for each group). Arrow and arrowhead indicate the administration of compound and glucose, respectively. *P < 0.05 (paired <i>t</i> test).</p

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of liver tissues.

A-D) Positively stained hepatocytes are observed in 3-month-old male (A) and female (B) TSOD mice and 9-month-old male (C) and female (D) db/db mice (original magnification ×400). E) The number of positively stained hepatocytes is significantly higher in 3-month-old male TSOD mice than in females (one-tailed t-test), but there is no significant sex difference in 9-month-old TSOD mice. F) For db/db mice, there is no significant sex difference in both age groups.</p

Serum FGF21 levels.

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