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Identification of a Molecular Activator for Insulin Receptor with Potent Anti-diabetic Effects*

Open AccessPublished:October 28, 2011DOI:https://doi.org/10.1074/jbc.M111.247387
      Insulin exerts its actions through the insulin receptor (IR) and plays an essential role in diabetes. The inconvenient daily injection and undesirable side-effects associated with insulin injection demand novel drugs for the diseases. To search for bioactive insulin mimetics, we developed an in vitro screening assay using phospho-IR ELISA. After screening the small molecule chemical libraries, we have obtained a compound (5,8-diacetyloxy-2,3-dichloro-1,4-naphthoquinone) that provokes IR activation by directly binding to the receptor kinase domain to trigger its kinase activity at micromolar concentrations. This compound selectively activates IR but not other receptors and sensitizes insulin's action. Moreover, it elevates glucose uptake in adipocytes and has oral hypoglycemic effect in wild-type C57BL/6J mice and db/db and ob/ob mice without demonstrable toxicity. Hence, this promising compound mimics the biological functions of insulin and is useful for further drug development for diabetes treatment.

      Introduction

      Type 2 diabetes mellitus is a heterogeneous disease that results from defective insulin actions and secretions. Various pharmacological agents are used to improve glucose homeostasis via different modes of action: sulfonylureas stimulate insulin secretion, biguanides (e.g. metformin) promote glucose utilization and reduce hepatic-glucose production, α-glucosidase inhibitors (e.g. acarbose) slow down carbohydrate absorption from the gut, and thiazolidinediones enhance cellular insulin action on glucose metabolism (
      • Fonseca V.A.
      • Kulkarni K.D.
      ). Insulin replacement therapy is also needed when insulin production declines in the patients with poor glycemic control (
      • Campbell R.K.
      ). In recent years, treatment strategies have focused on the development of novel therapeutic options to substitute insulin therapy. Several small compounds like demethylasterriquinone-B1 and TLK19780 have been identified as the functional insulin mimetics; however, they have poor bioavailability or low receptor specificity (
      • Pender C.
      • Goldfine I.D.
      • Manchem V.P.
      • Evans J.L.
      • Spevak W.R.
      • Shi S.
      • Rao S.
      • Bajjalieh S.
      • Maddux B.A.
      • Youngren J.F.
      ,
      • Zhang B.
      • Salituro G.
      • Szalkowski D.
      • Li Z.
      • Zhang Y.
      • Royo I.
      • Vilella D.
      • Díez M.T.
      • Pelaez F.
      • Ruby C.
      • Kendall R.L.
      • Mao X.
      • Griffin P.
      • Calaycay J.
      • Zierath J.R.
      • Heck J.V.
      • Smith R.G.
      • Moller D.E.
      ,
      • Wilkie N.
      • Wingrove P.B.
      • Bilsland J.G.
      • Young L.
      • Harper S.J.
      • Hefti F.
      • Ellis S.
      • Pollack S.J.
      ). Therefore, the search for new orally active insulin mimetics with stringent receptor selectivity is highly warranted.
      In this report, we demonstrate that the naphthoquinone derivative 5,8-diacetyloxy-2,3-dichloro-1,4-naphthoquinone (DDN)
      The abbreviations used are: DDN
      5,8-diacetyloxy-2,3-dichloro-1,4-naphthoquinone
      IRKT
      intracellular kinase domain of IR
      MEF
      mouse embryonic fibroblast
      IGF-1R
      insulin-like growth factor-1 receptor
      CSN
      2,3-bismethylsulfanyl-1,4-naphthoquinone
      2NH2-NQ
      2-amino-1,4-naphthoquinone
      ATPγS
      adenosine 5′-O-(thiotriphosphate)
      IRS-1
      insulin receptor substrate 1.
      is a highly selective IR activator that interacts directly with the IR tyrosine kinase domain to induce Akt and ERK phosphorylations. It is also an insulin sensitizer that enhances insulin's action to stimulate glucose uptake. Oral administration of this compound robustly decreases blood glucose in wild-type and diabetic ob/ob and db/db mice. Therefore, DDN is a bioactive insulin mimetic with hypoglycemic activity.

      DISCUSSION

      In this report, we have identified the 1,4-naphthoquinone derivative DDN as a new small molecular IR activator in vitro and in vivo. DDN has a simple chemical structure with prominent effect in selectively provoking IR activation and lowering blood glucose in animals. Thus, it represents a novel prototype for further chemical modification to generate a powerful therapeutic drug for diabetes.
      Zhang et al. have reported that DAQ B1 is an orally active IR ligand with anti-diabetic activity (
      • Zhang B.
      • Salituro G.
      • Szalkowski D.
      • Li Z.
      • Zhang Y.
      • Royo I.
      • Vilella D.
      • Díez M.T.
      • Pelaez F.
      • Ruby C.
      • Kendall R.L.
      • Mao X.
      • Griffin P.
      • Calaycay J.
      • Zierath J.R.
      • Heck J.V.
      • Smith R.G.
      • Moller D.E.
      ). Webster and his colleagues have conducted an extensive structure-activity relationship study on DAQ B1 derivatives and successfully identified the mono-indolyl-dihydroxybenzoquinones ZL-196 and LD-17, which activate IR signaling and effectively lower blood glucose in db/db mice (
      • Lin B.
      • Li Z.
      • Park K.
      • Deng L.
      • Pai A.
      • Zhong L.
      • Pirrung M.C.
      • Webster N.J.
      ). However, these compounds cannot selectively induce IR, and they also provoke other receptor tyrosine kinases, including IGF-1R, NGFR, and EGFR (
      • Lin B.
      • Li Z.
      • Park K.
      • Deng L.
      • Pai A.
      • Zhong L.
      • Pirrung M.C.
      • Webster N.J.
      ,
      • Liu K.
      • Xu L.
      • Szalkowski D.
      • Li Z.
      • Ding V.
      • Kwei G.
      • Huskey S.
      • Moller D.E.
      • Heck J.V.
      • Zhang B.B.
      • Jones A.B.
      ). In contrast, DDN specifically activates IR and its downstream cascades. A few classes of non-peptidyl IR activators have also been reported with different modes of actions. For example, TLK19780 is an insulin sensitizer, which potentiates insulin-triggered IR phosphorylation (
      • Pender C.
      • Goldfine I.D.
      • Manchem V.P.
      • Evans J.L.
      • Spevak W.R.
      • Shi S.
      • Rao S.
      • Bajjalieh S.
      • Maddux B.A.
      • Youngren J.F.
      ,
      • Manchem V.P.
      • Goldfine I.D.
      • Kohanski R.A.
      • Cristobal C.P.
      • Lum R.T.
      • Schow S.R.
      • Shi S.
      • Spevak W.R.
      • Laborde E.
      • Toavs D.K.
      • Villar H.O.
      • Wick M.M.
      • Kozlowski M.R.
      ). However, it is inactive when administrated alone. We also found that DDN potentiates the action of insulin in promoting IR activation and up-regulating glucose uptake. This additive effect might be a result of differential usage of IR ligand binding sites for insulin and DDN. Indeed, we have shown that DDN does not complete with insulin for IR binding but binds to the IR kinase domain directly. Hence, IR could simultaneously interact with both insulin on its extracellular domain and DDN on its intracellular kinase domain.
      When administrated orally, DDN has hypoglycemic function in both normal and diabetic mice models, and the glucose-lowering effect could be observed after 1-h administration. It is possible that the metabolites of DDN, in addition to DDN itself, might also possess the hypoglycemic activity in vivo. However, our cell-based in vitro studies showed that DDN directly bound IR and activated it within 5–15 min, suggesting DDN per se has a significant role in provoking IR activation. Moreover, IR activation in liver and muscle could be observed in 5 min, when DDN was injected into the bloodstream directly through the vena cava. It is interesting to note that DDN takes shorter time to reduce the blood glucose level in db/db mice than in normal C57BL/6 mice. One of the possible explanations is that the db/db mice have more adipose tissue than the normal mice, which may provide more insulin-responsive “storage sites” for the blood glucose. Thus, the db/db mice have more “targets” upon which DDN can exert its hypoglycemic function. Another possibility is that db/db mice have higher intestinal permeability (
      • Brun P.
      • Castagliuolo I.
      • Di Leo V.
      • Buda A.
      • Pinzani M.
      • Palù G.
      • Martines D.
      ). Because DDN is given via oral administration in the current studies, the higher absorption in the intestine of db/db mice thus promotes more DDN into the bloodstream, leading to a better efficacy in lowering blood glucose. It is noteworthy that a similar observation is made in the study using another quinone-based insulin mimetic Compound 2h. Compound 2h is more potent in lowing blood glucose in db/db mice than the lean control, suggesting that it may be a common characteristic for quinone-based insulin mimetics (
      • Liu K.
      • Xu L.
      • Szalkowski D.
      • Li Z.
      • Ding V.
      • Kwei G.
      • Huskey S.
      • Moller D.E.
      • Heck J.V.
      • Zhang B.B.
      • Jones A.B.
      ).
      CSN is also an effective IR activator in vitro. However, it is lethal to animals when administrated in vivo. 1,4-Naphthoquinones are widely distributed phenolic compounds in nature, and they display diverse pharmacological properties like antibacterial, antifungal, antiviral, anti-inflammatory, and antipyretic properties, including anticancer activity (
      • Kim B.H.
      • Yoo J.
      • Park S.H.
      • Jung J.K.
      • Cho H.
      • Chung Y.
      ,
      • Babula P.
      • Adam V.
      • Havel L.
      • Kizek R.
      ). Most of these quinoids belong to DNA-intercalating agents (
      • Yamashita N.
      • Maruyama M.
      • Yamazaki K.
      • Hamazaki T.
      • Yano S.
      ,
      • Prasad V.S.
      • Devi P.U.
      • Rao B.S.
      • Kamath R.
      ). For instance, Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), derived from Plumbagineae and Droseraceae families, can induce mammalian topoisomerase II-mediated DNA cleavage in vitro (
      • Fujii N.
      • Yamashita Y.
      • Arima Y.
      • Nagashima M.
      • Nakano H.
      ). Therefore, they are effective anticancer agents against murine fibrosarcoma, P388 lymphocytic leukemia, and A549 non-small cell lung cancer cells (
      • Krishnaswamy M.
      • Purushothaman K.K.
      ,
      • Singh U.V.
      • Udupa N.
      ,
      • Hsu Y.L.
      • Cho C.Y.
      • Kuo P.L.
      • Huang Y.T.
      • Lin C.C.
      ). In addition, the semiquinone radicals, generated either by one-electron reduction or two-electron reduction followed by a subsequent oxidation from quinine by dehydrogenase, damage the thiol groups or nucleophilic moieties of proteins (
      • Babich H.
      • Stern A.
      • Munday R.
      ). The oxidative stress induced by these quinone derivatives has been proposed to be responsible for initiation of cellular damage (
      • Smith M.T.
      ). Conceivably, the metabolites of CSN may covalently modify the thiol groups in many proteins, which may lead to its adverse side-effects. In contrast, DDN does not exhibit any intolerable side-effects when administrated to animals for 2 weeks, suggesting DDN and CSN might behave very differently.

      Acknowledgments

      We are indebted to Dr. Nicholas Webster at the University of California, San Diego for CHO-IR and CHO-IGF-1R stable cell lines.

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