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ENDOCRINE AND DIABETES

Identification of Novel Orally Available Small Molecule Insulin Mimetics

Veterans Affairs San Diego Healthcare System and the Veterans Medical Research Foundation, San Diego, California (B.L., N.J.G.W.); Department of Chemistry, University of California, Riverside, California (Z.L., K.P., L.D., M.C.P.); and Moore's Cancer Center and the Department of Medicine, University of California at San Diego, La Jolla, California (A.P., L.Z., N.J.G.W.)

Received for publication May 22, 2007
Accepted August 7, 2007.

	Abstract

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Oral hypoglycemic agents have great potential for the treatment of both type 1 and type 2 diabetes. Here we report the identification of novel, small-molecule, insulin mimetics that activate the insulin receptor (IR) in vivo and in vitro, stimulate the Akt and extracellular signal-regulated kinase pathways downstream of the IR, and mimic the ability of insulin to stimulate glucose uptake, glycogen synthesis, and lipid synthesis in 3T3-L1 adipocytes. However, the compounds do not mimic the mitogenic effect of insulin. In animals, these compounds have oral hypoglycemic effects in both normal C57BL6 mice and diabetic db/db mice. Quantitative structure activity relationship modeling on data from a library of 60 compounds has highlighted structural features that are important for IR agonist activity that can be used to guide design of second and third generation compounds with greater potency and specificity.

A second generation IR activator (Cpd.2) was subsequently identified with slightly greater potency and higher specificity (Liu et al., 2000; Wood et al., 2000). This compound has good pharmacokinetics in preclinical studies in rats, dogs, and monkeys. The addition of Cpd.2 to food prevents increased food intake and weight gain when normal mice are placed on a high fat diet for 7 weeks (Air et al., 2002; Strowski et al., 2004). No toxic effects were observed in treated mice, and Cpd.2 prevented lipid accumulation in the liver. Based on these findings, it was proposed that the hypothalamic effect of the insulin receptor activator over-rides its anabolic effect in the periphery.

In the course of investigating the binding of DAQ-B1 to the IR, we identified novel, small-molecule, insulin mimetics that have a simple mono-indolyl-dihydroxybenzoquinone structure. These molecules are cell-permeable, activate the IR and insulin signaling in intact cells, and stimulate glucose uptake and glycogen and lipid synthesis in adipocytes. Oral dosing of these compounds lowers blood glucose in normal and diabetic mice.

	Materials and Methods

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Materials. Antibodies to phospho-Akt(Ser473), phospho-ERK-(Thr202/Tyr204), ERK1/2, phospho-IR/IGF-IR(Tyr1162/Tyr1163), and phospho-EGFR(Tyr1173) were purchased from Cell Signaling Technologies (Worcester, MA), Upstate Biotechnologies Inc. (Charlottesville, VA), or BioSource (Camarillo, CA). Recombinant IR kinase domain (GST-IRK) was purchased from Calbiochem (San Diego, CA). Dulbecco's modified Eagle's/F-12, Ham's F-12, and minimal essential-Earle's culture media, GlutaMAX, fetal calf serum, and antibiotics were purchased from Invitrogen (Carlsbad, CA). The synthesis of the compounds in this article and the maintenance of the hIRcB, CHO-IR, CHO-IGF-IR, NIH3T3-EGFR, and 3T3-L1 cells have been described previously (Kayali et al., 2000; Eichhorn et al., 2002; Pirrung et al., 2002a).

ELISA for Activated Insulin Receptor. CHO-IR cells were cultured in 96-well plates, rendered quiescent by serum starvation for 24 h, and then stimulated with compounds for 10 min at 37°C. Compounds were dissolved in dimethyl sulfoxide and used at a final concentration of 30 µM in KRP-HEPES supplemented with 1 mM ascorbic acid. Control wells received vehicle or 8.3 nM insulin. Cells were lysed, and phosphorylated receptors were captured with antiphosphotyrosine antibodies (pY20) immobilized on ELISA plates. Bound receptors were washed then detected using an anti-IR antibody, followed by an horseradish peroxidase-conjugated secondary antibody and colorimetric detection with 3,3',5,5'-tetramethylbenzidine. Quantitative structure activity relationship (QSAR) calculations were performed using the TSAR 3.0 suite of programs (Accelrys, San Diego, CA). An explanation of the QSAR approach is given in Supplemental Methods and Results.

Immunoblotting. Cells were serum-starved for 72 h in 12-well plates and then stimulated with increasing concentrations of insulin or ZL-196 and LD-17 in KRP-HEPES supplemented with 1 mM ascorbic acid for 10 min at 37°C. Whole-cell extracts were immunoblotted as described previously (Webster et al., 2003).

Stimulation of Purified IRs and GST-IRK in Vitro. Insulin receptors were purified from hIRcB and CHO-IR cells, and the kinase assay was performed according to published procedures, with the exception that glycerol was omitted from the elution buffer (Resnik et al., 1998). In brief, equal amounts of IR were incubated with increasing concentrations of ZL-196 or LD-17 or insulin (8.3 nM) overnight at 4°C in kinase buffer. The phosphorylation reaction was initiated by the addition of ATP for 15 min at room temperature. The reaction was stopped, and tyrosine-phosphorylated IR was visualized by immunoblotting.

GST-IRK was incubated with increasing concentrations of ZL-196 or LD-17 for 30 min at room temperature in 50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, and 1 mM dithiothreitol. Phosphorylation was initiated by the addition of 50 µM ATP for 15 min at room temperature. Phosphorylated GST-IRK was visualized by immunoblotting.

2-Deoxyglucose Transport, Glucose Incorporation into Glycogen, and Lipid and Thymidine Incorporation into DNA. Biological assays were performed using standard procedures as described previously (Kayali et al., 1998; Eichhorn et al., 2002; Webster et al., 2003).

Oral Administration to Mice. Food was removed from cages 6 h before the experiment. Animals had free access to water throughout the study. Blood glucose levels were measured by tail venipuncture using a Hemocue glucose meter (Mission Viejo, CA). DAQ-B1, ZL-196, and LD-17 were given to C57BL6 and db/db mice by oral gavage in 0.5% methylcellulose. Blood glucose levels were measured at hourly intervals for C57BL6 mice or 2-h intervals for db/db mice over 6 h. All procedures were approved by the UCSD Animal Subjects Committee and conform to Institute of Laboratory Animal Resources (1996).

Statistical Analysis. Multiple group comparisons were performed by one-way analysis of variance with a Tukey post-test. Pairwise comparisons were performed using Student's t test with unequal variances.

	Results

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Mono-indolyl-dihydroxybenzoquinones Activate the IR in Whole Cells. The pseudosymmetrical bis-indolyl-quinone structure of DAQ-B1 suggested that the binding of this compound to the IR might be bipartite. Therefore, we synthesized two mono-indolyl-quinones, ZL-196 and ZL-202, as potential competitive inhibitors of DAQ-B1 (Fig. 1A). hIRcB cells were stimulated with insulin (17 nM) or DAQ-B1 (30 µM) in the presence of a 10-fold excess of ZL-196 or ZL-202 and whole-cell extracts immunoblotted for the phosphorylated IR. ZL-196 did not act as an inhibitor but, to our surprise, activated the IR (Fig. 1B). The complementary compound ZL-202 was inactive (Fig. 1C). This suggested that the active moiety in DAQ-B1 was the 7-prenylindole-dihydroxybenzoquinone.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Mono-indolyl-quinones activate the insulin receptor. A, structures of the two mono-indolyl-dihydroxybenzoquinones derived from DAQ-B1. B and C, hIRcB cells were treated with insulin (50 ng/ml), DAQ-B1 (30 µM), ZL-196 (300 µM), DAQ-B1 plus ZL-196, ZL-202 (300 µM), DAQ-B1 plus ZL-202, or vehicle for 10 min. Cell extracts were immunoblotted for phospho-IR(Tyr1162/Tyr1163), then stripped and reblotted for ERK1/2 or IR.

We then synthesized a panel of 60 mono-indolyl-dihydroxybenzoquinones (Pirrung et al., 2001, 2002a). Aliphatic, aromatic, and halogen substitution was tested at different positions on the indole ring, and the core quinone was also varied (Supplemental Table S1). This panel of compounds was tested by ELISA that detects the phosphorylated IR. Many of the compounds activated the IR in intact cells (Table 1). Qualitative inspection of the data suggests that substitution on the indole 6- or 7-position is associated with higher IR activation. To obtain a more quantitative assessment, we performed QSAR modeling on the IR data using multiple-regression and forward-feed neural networks. The generic structure of the mono-indolyl-quinone library is shown (Fig. 2A). For the multiple regression analysis, we considered theoretical, empirical, and whole-molecule parameters separately. The resulting model for IR activation using theoretical parameters shows a high correlation between predicted and measured activity and explains 94% of the variance in the data (R² = 0.9369; Fig. 2B). The empirical and whole-molecule parameters gave less predictive models (R² = 0.7637 and 0.4398, respectively; Supplemental Table S2). The significant individual terms in the theoretical parameter model involve substituents 6 and 7.

View this table: [in this window] [in a new window]   TABLE 1 Effect of monoquinones on IR activation by ELISA Data presented as percentage activity compared to insulin (17 nM), all compounds (Cpd.) used at 30 µM.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Quantitative structure activity relationship modeling. Compounds were used at 30 µM in ELISA. Insulin receptor activation was modeled using the TSAR program. A, generic structure of the compound library. Numbers indicate the sites of substitution considered. B, multiple linear regression model using theoretical substituent parameters. Activity is plotted on a scale of 0 to 1 with 1 being equivalent to 100% of the effect of insulin. Cross-validation by leaving out each of three groups in turn was performed to test predictive power for the multiple regression. C, forward feed neural network for theoretical substituent parameters. Predicted activity is plotted against actual activity measured by ELISA. Omission of 10% of the data and testing the root mean square fit to the model was performed to test predictive power after each cycle of neural network modeling. D, positions of substitutions that enhance IR activation are shown. E, structures of ZL-196 and LD-17 that were chosen for further study.

The forward feed neural network model using the theoretical parameters showed a higher correlation than the linear regression models (R² = 0.965; Fig. 2B). Here again the model using the theoretical parameters gave a more predictive model than either the empirical or whole-molecule parameter model (0.900 and 0.7986, respectively; Supplemental Table S3). The plot of actual versus predicted activity is shown in Fig. 2C. The parameters showing the greatest dependencies involved substituents 6 and 7 (Supplemental Table S4). Therefore, both QSAR models point to substituents 6 and 7 on the indole ring particularly important for the activation of the IR, in agreement with our earlier qualitative prediction (Fig. 2D). Two compounds, ZL-196 and LD-17, were chosen for further study that both contain large aliphatic or aromatic substitutions on position 7 of the indole ring (Fig. 2E).

Mono-indolyl-dihydroxybenzoquinones Are Direct and Selective IR Activators. The activity of a representative panel of 12 mono-indolyl-quinones compounds was confirmed by immunoblotting whole-cell extracts (Fig. 3A). In agreement with the ELISA, LD-17 was able to stimulate tyrosine phosphorylation of the IR. The compounds were also able to stimulate downstream signaling as Akt and ERK phosphorylation was increased (Fig. 3A). Interestingly, some compounds (e.g., LD-20B) activated ERK but did not appear to activate the IR, suggesting that there may be other tyrosine kinase targets in the cells. Dose-response studies were performed. Compounds ZL-196 and LD-17 caused a dose-dependent increase in the phosphorylation of IR, Akt, and ERK (Fig. 3B).

View larger version (63K): [in this window] [in a new window]   Fig. 3. Mono-indolyl-dihydroxybenzoquinones activate the IR directly and stimulate insulin signaling. A, hIRcB cells were treated with insulin (8.3 nM) or a series of monoquinones (30 µM) for 10 min and immunoblotted for phospho-IR(Tyr1162/Tyr1163), phospho-Akt(Ser473), or phospho-ERK-(Thr202/Tyr204). Blots were stripped and reblotted for ERK1/2. B, cells were treated with insulin (0.2, 1.7, and 8.3 nM), DAQ-B1, ZL-196, or LD-17 (3, 10, 30, and 100 µM) for 10 min. C, equal numbers of purified A-isoform or B-isoform IR receptors were stimulated in vitro with insulin (8.3 nM), ZL-196, or LD-17 (3, 10, and 30 µM) and then immunoblotted for phospho-IR(Tyr1162/Tyr1163). D, GST-IRK fusion protein was incubated with ZL-196 or LD-17 (0, 3, 10, 30, and 100 µM) in the presence of ATP and then immunoblotted for phospho-IR(Tyr1162/Tyr1163). E, CHO-IGF-IR cells were stimulated with IGF-I (0.2, 1.7, and 8.3 nM), DAQ-B1, ZL-196, or LD-17 (3, 10, 30, and 100 µM), or vehicle, and then immunoblotted for phospho-IGF-IR(Tyr1135/Tyr1136). F, NIH3T3-EGFR cells were stimulated with EGF (2, 4.2, and 8.3 nM), DAQ-B1, ZL-196, or LD-17 (10, 30, and 100 µM), or vehicle and then immunoblotted for phospho-EGFR(Tyr992). G, quantification of IR, IGF-IR, and EGFR activation from multiple experiments (n = 6, 4, and 3 for IR, IGF-IR, and EGFR, respectively). Data are presented in a separate panel for each compound. Phosphorylation of each receptor is normalized to 8.3 nM insulin, IGF-I, or EGF. *, statistical significance (p < 0.05) versus untreated value.

It is important to show that the IR is a direct target for these compounds. Therefore, we tested ZL-196 and LD-17 in vitro using purified insulin receptors, both A and B isoforms. ZL-196 and LD-17 activated both insulin receptor isoforms at 3 to 30 µM (Fig. 3C). In all cases, we observed activation of the IR at concentrations slightly below those required for activation of the receptor in whole cells.

We also tested whether the two new mimetics, ZL-196 and LD-17, could activate a recombinant IR kinase domain. A GST fusion protein containing the IR kinase domain was treated with increasing concentrations of ZL-196 and LD-17 in vitro and then allowed to autophosphorylate in the presence of ATP. Both ZL-196 and LD-17 caused a dose-dependent increase in autophosphorylation, which was maximal at 100 µM, suggesting that activation is less efficient on the monomeric IRK than on the heterotetrameric IR (Fig. 3D).

The selectivity of these compounds for the IR was tested by stimulating CHO-IGF-IR or NIH3T3-EGFR cells and then immunoblotting extracts for the phosphorylated form of the respective receptor (Fig. 3, E and F). DAQ-B1 and ZL-196 caused a dose-dependent increase in phosphorylation of the IR and IGF-IR but were less effective at activating the EGFR (Fig. 3G). LD-17 was a strong activator of the IR and a weaker activator of the IGF-IR and EGFR (Fig. 3G).

Mono-indolyl-dihydroxybenzoquinones Mimic Biological Effects of Insulin. At the cellular level, ZL-196, LD-17, and DAQ-B1 cause a dose-dependent increase in glucose transport in 3T3-L1 adipocytes (Fig. 4A). The maximal stimulation of transport by the compounds was only 50% of the effect of insulin. Two compounds, ZL-197 and ZL-199, which do not activate the IR, were unable to stimulate glucose transport (data not shown). The addition of insulin in the presence of a maximal dose of compound caused a further increase to the level seen with insulin alone, indicating that the compounds do not interfere with the ability of insulin to stimulate transport or increase the maximal effect. Treatment with the compounds did not alter the total levels of GLUT4 or GLUT1 during the 30-min incubation (Fig. 4B). ZL-196 and LD-17 stimulated glucose incorporation into glycogen (Fig. 4C) and lipid (Fig. 4D) to the same degree as insulin. A negative control, compound ZL-202, which does not activate the IR, was unable to stimulate glucose incorporation into either glycogen or lipid. DAQ-B1 caused a decrease in glycogen content due to an unexpected glycogenolytic effect of this compound (Supplemental Fig. 1). Interestingly, none of the insulin mimetic compounds stimulated thymidine incorporation in hIRcB cells (Fig. 4E). This may suggest that the compounds mimic the metabolic but not the mitogenic effects of insulin, as had been suggested previously (Air et al., 2002).

View larger version (32K): [in this window] [in a new window]   Fig. 4. Biological effects of ZL-196 and LD-17. A, 3T3-L1 adipocytes were stimulated with insulin (0.2, 1.7, and 8.3 nM) or compounds (1, 10, 30, and 100 µM) for 30 min. Transport was performed for 10 min with [3H]2-deoxyglucose (2-DOG). B, 3T3-L1 adipocytes were stimulated with insulin (8.3 nM) or compounds (100 µM) for 30 min. Whole-cell extracts were immunoblotted for GLUT1, GLUT4, or tubulin. C, 3T3-L1 adipocytes were stimulated with insulin (0.2, 1.7, and 8.3 nM) or compounds (100 µM) for 2 h in the presence of 14C-glucose. Glycogen was extracted, and precipitated counts were measured. D, 3T3-L1 adipocytes were stimulated with insulin (0.2, 1.7, and 8.3 nM) or compounds (100 µM) for 2 h in the presence of 14C-glucose. Lipids were extracted, and incorporated counts were measured. E, hIRcB cells were stimulated with insulin (8.3 nM) or compounds (30 and 100 µM) for 16 h. [3H]thymidine was added for the last 4 h. Cells were washed, and incorporated counts were measured. * indicate statistical significance (P < 0.05) versus untreated cells.

View larger version (30K): [in this window] [in a new window]   Fig. 5. Hypoglycemic effects in vivo. A, acute hypoglycemic effect in normal mice. C57BL6 mice (n = 6) were starved for 6 h and then dosed with DAQ-B1, ZL-196, or LD-17 (20 mg/kg) as a suspension in 0.5% methylcellulose by oral gavage. Mean basal glucose level was 170 mg/dl. B, chronic hypoglycemic effect of compounds in diabetic mice. db/db mice (n = 6) were starved for 6 h and then dosed with DAQ-B1, ZL-196, or LD-17 (20 mg/kg) as a suspension in 0.5% methylcellulose by oral gavage. Mean fasting glucose level in the db/db mice was 294 mg/dl. C and D, db/db mice were dosed with ZL-196 or LD-17 at 5, 10, or 20 mg/kg by oral gavage. For all panels, blood glucose levels are presented as percentage vehicle-treated mice. * indicate statistical significance (p < 0.05) versus basal value at t = 0.

We also tested the compounds in the db/db model of type 2 diabetes. Administration of ZL-196, LD-17, or DAQ-B1 as a 20 mg/kg oral suspension caused a steady decline in blood glucose levels over 6 h in the db/db diabetic mice (Fig. 5B). The observed hypoglycemic effect of DAQ-B1 in this model agrees with a previous report (Zhang et al., 1999). Dose-response studies with ZL-196 (Fig. 5C) and LD-17 (Fig. 5D) demonstrated a dose-dependent decrease in blood glucose. Thus, the new insulin mimetics have hypoglycemic effects in both normal and diabetic mice.

	Discussion

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This article identifies a new class of small-molecule IR activators. This class of compounds contains 6- or 7-substituted indoles attached to dihydroxybenzoquinone. The structure is related to, but simpler than, the asterriquinone class of IR activators (Zhang et al., 1999). Our finding that the 7-prenyl-indole ring is the active substructure of DAQ-B1 confirms our previous conclusion from a methylation-scanning study of the parental compound. In that study, we found that methyl substitution on the 7-prenyl-indole ring was not tolerated; however, methyl substitution on the 2 isoprenylindole ring did not alter activity (Pirrung et al., 2005). We concluded that the 7-prenyl-indole, but not the 2-isoprenylindole, probably makes close contacts with the insulin receptor kinase domain.

This new class of IR activators offers distinct advantages over the asterriquinones. At a practical level, monosubstitution on the dihydroxyquinone core is a facile reaction, and we have previously published high-efficiency synthetic routes to these molecules (Pirrung et al., 2001, 2002a). The problems with regioselectivity that were encountered in the synthesis of the disubstituted parental compound DAQ-B1 are eliminated (Pirrung et al., 2002b). The asterriquinones also have limited cell permeability (Kaji et al., 1998). An earlier study demonstrated that exposure of P388 leukemia cells to 30 µM DAQ-A1 for 1 h resulted in an intracellular concentration of only 60 pM (Kaji et al., 1998). Its dimethyl derivative, AQ-A1, showed an increased intracellular concentration of 200 pM; however, the mono-methyl derivative showed an intracellular concentration of only 70 pM. This is consistent with our finding that activation of the IR in vitro is more potent than activation in whole cells. Lipinksi's Rules [<5 H-bond donors (sum of OHs and NHs); molecular mass, <500 Da; log P <5; <10 H-bond acceptors (sum of Ns and Os)] can be used as a guide to predict cell permeability (Lipinski et al., 2001). The compound DAQ-B1 has four H-bond donors (molecular mass, 506 Da) and two H-bond acceptors, so the lack of permeation is not related to these molecular properties. Log P is an indicator of the lipophilic properties of a compound, and values >5 are predictive of poor cell permeability. The calculated log P for DAQ-B1 is 6.96; Cpd.2 has a log P of 3.57 and consequently increased activity in vivo. The log P values for ZL-196 and LD-17 are 2.65 and 1.97, respectively; thus, they should have improved cellular uptake.

At the cellular level, mono-indolyl-dihydroxybenzoquinones are metabolic insulin mimetics. They stimulate glucose uptake and its incorporation into glycogen and lipid, whereas DAQ-B1 only stimulates glucose uptake in 3T3-L1 cells. This highlights a marked difference between the mono-indolyl-quinones described here and DAQ-B1. In mice, the mono-indolyl-quinones have a glucose-lowering effect that is comparable to DAQ-B1. It has been reported that Cpd.2 stimulates glucose uptake and glycogen synthesis and suppresses lipolysis in rat adipocytes (Ding et al., 2002). We have tested two separate preparations of Cpd.2, one synthesized in our laboratory and the other commercial. Cpd.2 at 30 µM has 60% the activity of 17 nM insulin in our ELISA, and the corresponding mono-indolyl-quinone (ZL-194) shows only 25% activity. These results are consistent with the partial effect of Cpd.2 on glucose transport (50%), glycogen synthesis (30%), and lipolysis (45%) (Liu et al., 2000; Al-Khalili et al., 2003). With the caveat that we have not performed a direct comparison of biological effects, our ELISA and biological data suggest that the new monoquinones are similar to Cpd.2.

In conclusion, we have identified a new class of 2,5-dihydroxy-3-(1H-indol-3-yl)-[1,4]-benzoquinone insulin receptor activators that have a hypoglycemic effect in mice. Whereas the micromolar concentrations of ZL-196 and LD-17 required to elicit biological effects precludes preclinical development, these concentrations are typical of "lead" compounds. We hope the rapid screening of potential structures in silico using our QSAR models, coupled with simple synthetic routes to mono-indolyl-benzoquinones, will allow the rapid identification of compounds containing modified indole groups and quinones that are more potent and more selective, with fewer off-target effects.

	Acknowledgements

 
The NIH3T3-EGFR cells were a generous gift from Dr. Frank Furnari [University of California at San Diego (UCSD)].

	Footnotes

 
This work was supported by the American Diabetes Association (to M.C.P.) and the National Institutes of Health (Grant R03 AG023191) (to N.J.G.W.). L.Z. was supported by a fellowship from Merck Pharmaceuticals. N.J.G.W. is a faculty member of the UCSD Biomedical Sciences Graduate Program. A.P. is a student at the UCSD School of Medicine.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.126102.

ABBREVIATIONS: DAQ-B1, demethylasterriquinone-B1, 2-[2-(1,1-dimethyl-allyl)-1H-indol-3-yl]-3,6-dihydroxy-5-[7-(3-methyl-but-2-enyl)-1H-indol-3-yl]-[1,4]benzoquinone; ZL-196, 2,5-dihydroxy-3-(7-(3-methyl-but-2-enyl)-1H-indol-3-yl)-[1,4]-benzoquinone; ZL-202, 2,5-dihydroxy-3-[2-(1,1-dimethyl-allyl)-1H-indol-3-yl]-[1,4]-benzoquinone; LD-17, 2,5-dihydroxy-3-(7-benzyloxy-1H-indol-3-yl)-[1,4]-benzoquinone; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; Cpd.2, 3,6-dihydroxy-2-(1-methyl-indol-3-yl)-5-phenyl-[1,4]-benzoquinone; QSAR, quantitative structure activity relationship; ELISA, enzyme-linked immunosorbent assay; IR, insulin receptor; IGF, insulin-like growth factor-1; CHO, Chinese hamster ovary; ERK, extracellular-regulated kinase; GST, glutathione transferase; hIRCB, rat 1 fibroblasts overexpressing the human IR; KRP, Krebs-Ringer phosphate; LD-20B, 2,5-dihydroxy-3-(5-methyl-1H-indol-3-yl)-[1,4]-benzoquinone; ZL-194, 2,5-dihydroxy-3-(N-methyl-indol-3-yl)-[1,4]-benzoquinone.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Nicholas J. G. Webster, Stein Clinical Research Bldg. 201, Department of Medicine (0673), University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0673. E-mail: nwebster{at}ucsd.edu

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