CDKN1C-mediated growth inhibition by an EZH1/2 dual inhibitor overcomes resistance of mantle cell lymphoma to ibrutinib.

Mantle cell lymphoma (MCL) is a rare subtype of non-Hodgkin's lymphoma, which is characterized by overexpression of cyclin D1. Although novel drugs, such as ibrutinib, show promising clinical outcomes, relapsed MCL often acquires drug resistance. Therefore, alternative approaches for refractory and relapsed MCL are needed. Here, we examined whether a novel inhibitor of enhancer of zeste homologs 1 and 2 (EZH1/2), OR-S1 (a close analog of the clinical-stage compound valemetostat), had an antitumor effect on MCL cells. In an ibrutinib-resistant MCL patient-derived xenograft (PDX) mouse model, OR-S1 treatment by oral administration significantly inhibited MCL tumor growth, whereas ibrutinib did not. In vitro growth assays showed that compared with an established EZH2-specific inhibitor GSK126, OR-S1 had a marked antitumor effect on MCL cell lines. Furthermore, comprehensive gene expression analysis was performed using OR-S1-sensitive or insensitive MCL cell lines and showed that OR-S1 treatment modulated B-cell activation, differentiation, and cell cycle. In addition, we identified Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, also known as p57, KIP2), which contributes to cell cycle arrest, as a direct target of EZH1/2 and showed that its expression influenced MCL cell proliferation. These results suggest that EZH1/2 may be a potential novel target for the treatment of aggressive ibrutinib-resistant MCL via CDKN1C-mediated cell cycle arrest.

Dual inhibition of enhancer of zeste homolog 1/2 overactivates WNT signaling to deplete cancer stem cells in multiple myeloma.

Multiple myeloma (MM) is an incurable hematological malignancy caused by accumulation of abnormal clonal plasma cells. Despite the recent development of novel therapies, relapse of MM eventually occurs as a result of a remaining population of drug-resistant myeloma stem cells. Side population (SP) cells show cancer stem cell-like characteristics in MM; thus, targeting these cells is a promising strategy to completely cure this malignancy. Herein, we showed that SP cells expressed higher levels of enhancer of zeste homolog (EZH) 1 and EZH2, which encode the catalytic subunits of Polycomb repressive complex 2 (PRC2), than non-SP cells, suggesting that EZH1 as well as EZH2 contributes to the stemness maintenance of the MM cells and that targeting both EZH1/2 is potentially a significant therapeutic approach for eradicating myeloma stem cells. A novel orally bioavailable EZH1/2 dual inhibitor, OR-S1, effectively eradicated SP cells and had a greater antitumor effect than a selective EZH2 inhibitor in vitro and in vivo, including a unique patient-derived xenograft model. Moreover, long-term continuous dosing of OR-S1 completely cured mice bearing orthotopic xenografts. Additionally, PRC2 directly regulated WNT signaling in MM, and overactivation of this signaling induced by dual inhibition of EZH1/2 eradicated myeloma stem cells and negatively affected tumorigenesis, suggesting that repression of WNT signaling by PRC2 plays an important role in stemness maintenance of MM cells. Our results show the role of EZH1/2 in the maintenance of myeloma stem cells and provide a preclinical rationale for therapeutic application of OR-S1, leading to significant advances in the treatment of MM.

Discovery of DS-6930, a potent selective PPARγ modulator. Part II: Lead optimization.

Attempts were made to reduce the lipophilicity of previously synthesized compound (II) for the avoidance of hepatotoxicity. The replacement of the left-hand side benzene with 2-pyridine resulted in the substantial loss of potency. Because poor membrane permeability was responsible for poor potency in vitro, the adjustment of lipophilicity was examined, which resulted in the discovery of dimethyl pyridine derivative (I, DS-6930). In preclinical studies, DS-6930 demonstrated high PPARγ agonist potency with robust plasma glucose reduction. DS-6930 maintained diminished PPARγ-related adverse effects upon toxicological evaluation in vivo, and demonstrated no hepatotoxicity. Cofactor recruitment assay showed that several cofactors, such as RIP140 and PGC1, were significantly recruited, whereas several canonical factors was not affected. This selective cofactor recruitment was caused due to the distinct binding mode of DS-6930. The calcium salt, DS-6930b, which is expected to be an effective inducer of insulin sensitization without edema, could be evaluated clinically in T2DM patients.

Discovery of DS-6930, a potent selective PPARγ modulator. Part I: Lead identification.

The lead identification of a novel potent selective PPARγ agonist, DS-6930 is reported. To avoid PPARγ-related adverse effects, a partial agonist was designed to prevent the direct interaction with helix 12 of PPARγ-LBD. Because the TZD group is known to interact with helix 12, the TZD in efatutazone (CS-7017) was replaced to discover novel PPARγ intermediate partial agonist 8i. The optimization of 8i yielded 13ac with high potency in vitro. Compound 13ac exhibited robust plasma glucose lowering effects comparable to those of rosiglitazone (3 mg/kg) in Zucker diabetic fatty rats. Upon toxicological evaluation, compound 13ac (300 mg/kg) induced hemodilution to a lower extent than rosiglitazone; however, 13ac elevated liver enzyme activities. X-ray crystallography revealed no direct interaction of 13ac with helix 12, and the additional lipophilic interactions are also suggested to be related to the maximum transcriptional activity of 13ac.

EZH1/2 dual inhibitors suppress HTLV-1-infected cell proliferation and hyperimmune response in HTLV-1-associated myelopathy.

Background: Human T-cell leukemia virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy (HAM), adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated uveitis, and pulmonary diseases. Although both HAM and ATL show proliferation of infected cells, their pathogeneses are quite different. In particular, the pathogenesis of HAM is characterized by hyperimmune responses to HTLV-1-infected cells. Recently, we demonstrated the overexpression of histone methyltransferase EZH2 in ATL cells and the cytotoxic effects of EZH2 inhibitors and EZH1/2 dual inhibitors on these cells. However, these phenomena have never been studied in HAM. Furthermore, what effect these agents have on the hyperimmune response seen in HAM is completely unknown. Methods: In this study, we investigated histone methyltransferase expression levels in infected cell populations (CD4+ and CD4+CCR4+ cells) from patients with HAM using microarray and RT-qPCR analyses. Next, using an assay system that utilizes the spontaneous proliferation characteristic of peripheral blood mononuclear cells derived from patients with HAM (HAM-PBMCs), we investigated the effects of EZH2 selective inhibitors (GSK126 and tazemetostat) and EZH1/2 dual inhibitors (OR-S1 and valemetostat, also known as DS-3201), particularly on cell proliferation rate, cytokine production, and HTLV-1 proviral load. We also examined the effect of EZH1/2 inhibitors on the proliferation of HTLV-1-infected cell lines (HCT-4 and HCT-5) derived from patients with HAM. Results: We found elevated expression of EZH2 in CD4+ and CD4+CCR4+ cells from patients with HAM. EZH2 selective inhibitors and EZH1/2 inhibitors significantly inhibited spontaneous proliferation of HAM-PBMC in a concentration-dependent manner. The effect was greater with EZH1/2 inhibitors. EZH1/2 inhibitors also reduced the frequencies of Ki67+ CD4+ T cells and Ki67+ CD8+ T cells. Furthermore, they reduced HTLV-1 proviral loads and increased IL-10 levels in culture supernatants but did not alter IFN-γ and TNF-α levels. These agents also caused a concentration-dependent inhibition of the proliferation of HTLV-1-infected cell lines derived from patients with HAM and increased annexin-V(+)7-aminoactinomycin D(-) early apoptotic cells. Conclusion: This study showed that EZH1/2 inhibitors suppress HTLV-1-infected cell proliferation through apoptosis and the hyperimmune response in HAM. This indicates that EZH1/2 inhibitors may be effective in treating HAM.

Substituents at the naphthalene C3 position of (-)-Cercosporamide derivatives significantly affect the maximal efficacy as PPARγ partial agonists.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a potential drug target for treating type 2 diabetes. The selective PPARγ modulators (SPPARMs), which partially activate the PPARγ transcriptional activity, are considered to improve the plasma glucose level with attenuated PPARγ related adverse effects. However, the relationships between desired pharmacological profiles and ligand specific PPARγ transcriptional profiles have been unclear. And there is also little knowledge of how to control ligand specific PPARγ transcriptional profiles. Herein, we present synthesis of novel derivatives containing substituent at naphthalene C3 position of compound 1. The novel derivatives showed various maximal efficacies as PPARγ partial agonist.

Altered glucose homeostasis in mice with liver-specific deletion of Src homology phosphatase 2.

The Src homology 2 domain-containing protein-tyrosine phosphatase Shp2 has been implicated in a variety of growth factor signaling pathways, but its role in insulin signaling has remained unresolved. In vitro studies suggest that Shp2 is both a negative and positive regulator of insulin signaling, although its physiological function in a number of peripheral insulin-responsive tissues remains unknown. To address the metabolic role of Shp2 in the liver, we generated mice with either chronic or acute hepatic Shp2 deletion using tissue-specific Cre-LoxP and adenoviral Cre approaches, respectively. We then analyzed insulin sensitivity, glucose tolerance, and insulin signaling in liver-specific Shp2-deficient and control mice. Mice with chronic Shp2 deletion exhibited improved insulin sensitivity and increased glucose tolerance compared with controls. Acute Shp2 deletion yielded comparable results, indicating that the observed metabolic effects are directly caused by the lack of Shp2 in the liver. These findings correlated with, and were most likely caused by, direct dephosphorylation of insulin receptor substrate (IRS)1/2 in the liver, accompanied by increased PI3K/Akt signaling. In contrast, insulin-induced ERK activation was dramatically attenuated, yet there was no effect on the putative ERK site on IRS1 (Ser(612)) or on S6 kinase 1 activity. These studies show that Shp2 is a negative regulator of hepatic insulin action, and its deletion enhances the activation of PI3K/Akt pathway downstream of the insulin receptor.

Pharmacology and in vitro profiling of a novel peroxisome proliferator-activated receptor γ ligand, Cerco-A.

Peroxisome proliferator-activated receptor γ (PPARγ; NR1C3) is known as a key regulator of adipocytogenesis and the molecular target of thiazolidinediones (TZDs), also known as antidiabetic agents. Despite the clinical benefits of TZDs, their use is often associated with adverse effects including peripheral edema, congestive heart failure, and weight gain. Here we report the identification and characterization of a non-thiazolidinedione PPARγ partial agonist, Cerco-A, which is a derivative of the natural product, (-)-cercosporamide. Cerco-A was found to be a binder of the PPARγ ligand-binding domain in a ligand competitive binding assay and showed a unique cofactor recruitment profile compared to rosiglitazone. A crystal structure analysis revealed that Cerco-A binds to PPARγ without direct hydrogen bonding to helix12. In PPARγ transcriptional activation assay and an adipocyte differentiation assay, Cerco-A was a potent partial agonist of PPARγ. After a 14-day oral administration, once per day of Cerco-A in Zucker diabetic fatty (ZDF) rats, an apparent decrease of plasma glucose and triglyceride was observed, as with pioglitazone. To evaluate drug safety, Cerco-A was administered for 13 days orally in non-diabetic Zucker fatty (ZF) rats. Each of the hemodilution parameters (hematocrit, red blood cells number, and hemoglobin), which are considered as undesirable effects of TZDs, was improved significantly compared to pioglitazone. While Cerco-A showed body weight gain, as with pioglitazone, Cerco-A had significantly lower effects on heart and white adipose tissues weight gain. The results suggest that Cerco-A offers beneficial effects on glycemic control with attenuated undesirable side effects.

Role of Rho-kinase in regulation of insulin action and glucose homeostasis.

Accumulating evidence indicates an important role for serine phosphorylation of IRS-1 in the regulation of insulin action. Recent studies suggest that Rho-kinase (ROK) is a mediator of insulin signaling, via interaction with IRS-1. Here we show that insulin stimulation of glucose transport is impaired when ROK is chemically or biologically inhibited in cultured adipocytes and myotubes and in isolated soleus muscle ex vivo. Inactivation of ROK also reduces insulin-stimulated IRS-1 tyrosine phosphorylation and PI3K activity. Moreover, inhibition of ROK activity in mice causes insulin resistance by reducing insulin-stimulated glucose uptake in skeletal muscle in vivo. Mass spectrometry analysis identifies IRS-1 Ser632/635 as substrates of ROK in vitro, and mutation of these sites inhibits insulin signaling. These results strongly suggest that ROK regulates insulin-stimulated glucose transport in vitro and in vivo. Thus, ROK is an important regulator of insulin signaling and glucose metabolism.

Discovery of a novel selective PPARgamma modulator from (-)-Cercosporamide derivatives.

In an investigation of (-)-Cercosporamide derivatives with a plasma glucose-lowering effect, we found that N-benzylcarboxamide derivative 4 was a partial agonist of PPARgamma. A SAR study of the substituents on carboxamide nitrogen afforded the N-(1-naphthyl)methylcarboxamide derivative 23 as the most potent selective PPARgamma modulator. An X-ray crystallography study revealed that compound 23 bounded to the PPARgamma ligand binding domain in a unique way without any interaction with helix12. Compound 23 displayed a potent plasma glucose-lowering effect in db/db mice without the undesirable increase in body fluid and heart weight that is typically observed when PPARgamma full agonists are administrated.

(-)-Cercosporamide derivatives as novel antihyperglycemic agents.

In our exploratory campaign for an antihyperglycemic agent with a novel mechanism of action, (-)-Cercosporamide 1, which is known as an antifungal agent, showed a potent plasma glucose lowering effect in hyperglycemic KK/Ta mice. The trouble was that it was accompanied by a decrease in food intake and a loss of body weight. We synthesized some (-)-Cercosporamide derivatives and succeeded to separate these actions. N,O-ketal type derivatives, especially compound 10, had the most potent plasma glucose lowering effect without affecting the food consumption or body weight.

Potent antidiabetic effects of rivoglitazone, a novel peroxisome proliferator-activated receptor-gamma agonist, in obese diabetic rodent models.

The pharmacological effects of rivoglitazone, a novel thiazolidinedione-derivative peroxisome proliferator-activated receptor (PPAR)-gamma agonist, were characterized in vitro and in vivo. Rivoglitazone activated human PPARgamma more potently compared with rosiglitazone and pioglitazone and had little effect on PPARalpha and PPARdelta activity in luciferase reporter assays. In Zucker diabetic fatty (ZDF) rats, 14-day administration of rivoglitazone decreased the plasma glucose and triglyceride (TG) levels in a dose-dependent manner. The glucose-lowering effect of rivoglitazone was much more potent than those of pioglitazone (ED(50): 0.19 vs. 34 mg/kg) and rosiglitazone (ED(50): 0.20 vs. 28 mg/kg). In addition, rivoglitazone showed potent antidiabetic effects in diabetic db/db mice. In Zucker fatty rats, rivoglitazone at a dose of 0.1 mg/kg clearly ameliorated insulin resistance and lowered plasma TG levels by accelerating the clearance of plasma TG. Gene expression analysis in the liver and heart of ZDF rats treated with rivoglitazone for 14 days suggested that rivoglitazone may reduce hepatic glucose production and modulate the balance of the cardiac glucose/fatty acid metabolism in diabetic animals. In summary, we showed that rivoglitazone is a potent and selective PPARgamma agonist and has a potent glucose-lowering effect via improvement of the insulin resistance in diabetic animal models.

Structure-Activity Relationship Studies of 3- or 4-Pyridine Derivatives of DS-6930.

Derivatization efforts were continued to discover backups for a potent selective PPARγ modulator, DS-6930. In this Letter, the replacement of 2-pyridine ring in DS-6930 with 3- or 4-pyridyl group is reported. As the introduction of substituents on the pyridine ring did not provide potent partial agonists, modifications of benzimidazole ring were explored to discover potent intermediate agonists. 4'-Alkoxy substituted benzimidazoles failed to show potent efficacy in vivo, whereas 7'-fluoro benzimidazole 3g (DS19161384) was found to result in robust plasma glucose reductions with excellent DMPK profiles.

Targeting Excessive EZH1 and EZH2 Activities for Abnormal Histone Methylation and Transcription Network in Malignant Lymphomas.

Although global H3K27me3 reprogramming is a hallmark of cancer, no effective therapeutic strategy for H3K27me3-high malignancies harboring EZH2WT/WT has yet been established. We explore epigenome and transcriptome in EZH2WT/WT and EZH2WT/Mu aggressive lymphomas and show that mutual interference and compensatory function of co-expressed EZH1 and EZH2 rearrange their own genome-wide distribution, thereby establishing restricted chromatin and gene expression signatures. Direct comparison of leading compounds introduces potency and a mechanism of action of the EZH1/2 dual inhibitor (valemetostat). The synthetic lethality is observed in all lymphoma models and primary adult T cell leukemia-lymphoma (ATL) cells. Opposing actions of EZH1/2-polycomb and SWI/SNF complexes are required for facultative heterochromatin formation. Inactivation of chromatin-associated genes (ARID1A, SMARCA4/BRG1, SMARCB1/SNF5, KDM6A/UTX, BAP1, KMT2D/MLL2) and oncovirus infection (HTLV-1, EBV) trigger EZH1/2 perturbation and H3K27me3 deposition. Our study provides the mechanism-based rationale for chemical dual targeting of EZH1/2 in cancer epigenome.

Direct comparison of in vivo antisense activity of ENA oligonucleotides targeting PTP1B mRNA with that of 2'-O-(2-methoxy)ethyl-modified oligonucleotides.

Protein-tyrosine phosphatase 1B (PTP1B) inhibitory activity of the 2'-O-(2-methoxy)ethyl (2'- MOE)-modified gapmer antisense oligonucleotide, ISIS113715, was previously reported. This antisense oligonucleotide increases insulin sensitivity and normalizes plasma glucose levels in diabetic ob/ob and db/db mice. In the present study, the isosequential 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified oligonucleotide, ENA-1, was synthesized, and its ability to further improve the downregulation of PTP1B in db/db mice was examined. We demonstrated that, compared with ISIS113715, intraperitoneal and subcutaneous administration of ENA-1 more effectively decreased the plasma glucose levels in db/db mice. Moreover, ENA-1 decreased expression of PTP1B in the liver and fat of db/db mice more effectively than ISIS113715. We describe for the first time the functional comparison of 2'-MOE- and ENA-modified antisense oligonucleotides. Our data indicate that the enhancement of the efficacy of antisense oligonucleotides by ENA modifications is superior to that of second-generation 2'-MOE modifications in certain aspects.

IDH2 and NPM1 Mutations Cooperate to Activate Hoxa9/Meis1 and Hypoxia Pathways in Acute Myeloid Leukemia.

IDH1 and IDH2 mutations occur frequently in acute myeloid leukemia (AML) and other cancers. The mutant isocitrate dehydrogenase (IDH) enzymes convert α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of AML in which mice were transplanted with nucleophosmin1 (NPM)(+/-) hematopoietic stem/progenitor cells cotransduced with four mutant genes (NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD), which often occur simultaneously in human AML patients. Conditional deletion of IDH2/R140Q blocked 2-HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. IDH2/R140Q was necessary for the engraftment or survival of NPMc(+) cells in vivo. Gene expression analysis indicated that NPMc increased expression of Hoxa9. IDH2/R140Q also increased the level of Meis1 and activated the hypoxia pathway in AML cells. IDH2/R140Q decreased the 5hmC modification and expression of some differentiation-inducing genes (Ebf1 and Spib). Taken together, our results indicated that IDH2 mutation is critical for the development and maintenance of AML stem-like cells, and they provided a preclinical justification for targeting mutant IDH enzymes as a strategy for anticancer therapy.

Regulation of glucose transport by ROCK1 differs from that of ROCK2 and is controlled by actin polymerization.

A role of Rho-associated coiled-coil-containing protein kinase (ROCK)1 in regulating whole-body glucose homeostasis has been reported. However, cell-autonomous effects of ROCK1 on insulin-dependent glucose transport in adipocytes and muscle cells have not been elucidated. To determine the specific role of ROCK1 in glucose transport directly, ROCK1 expression in 3T3-L1 adipocytes and L6 myoblasts was biologically modulated. Here, we show that small interfering RNA-mediated ROCK1 depletion decreased insulin-induced glucose transport in adipocytes and myoblasts, whereas adenovirus-mediated ROCK1 expression increased this in a dose-dependent manner, indicating that ROCK1 is permissive for glucose transport. Inhibition of ROCK1 also impaired glucose transporter 4 translocation in 3T3-L1 adipocytes. Importantly, the ED50 of insulin for adipocyte glucose transport was reduced when ROCK1 was expressed, leading to hypersensitivity to insulin. These effects are dependent on actin cytoskeleton remodeling, because inhibitors of actin polymerization significantly decreased ROCK1's effect to promote insulin-stimulated glucose transport. Unlike ROCK2, ROCK1 binding to insulin receptor substrate (IRS)-1 was not detected by immunoprecipitation, although cell fractionation demonstrated both ROCK isoforms localize with IRS-1 in low-density microsomes. Moreover, insulin's ability to increase IRS-1 tyrosine 612 and serine 632/635 phosphorylation was attenuated by ROCK1 suppression. Replacing IRS-1 serine 632/635 with alanine reduced insulin-stimulated phosphatidylinositol 3-kinase activation and glucose transport in 3T3-L1 adipocytes, indicating that phosphorylation of these serine residues of IRS-1, which are substrates of the ROCK2 isoform in vitro, are crucial for maximal stimulation of glucose transport by insulin. Our studies identify ROCK1 as an important positive regulator of insulin action on glucose transport in adipocytes and muscle cells.

Synthesis and biological evaluation of novel (-)-Cercosporamide derivatives as potent selective PPARγ modulators.

Selective peroxisome proliferator-activated receptor gamma (PPARγ) modulators are expected to be a novel class of drugs improving plasma glucose levels without PPARγ-related adverse effects. As a continuation of our studies for (-)-Cercosporamide derivatives as selective PPARγ modulators, we synthesized substituted naphthalene type compounds and identified the most potent compound 15 (EC(50) = 0.94 nM, E(max) = 38%). Compound 15 selectively activated PPARγ transcription and did not activate PPARα and PPARδ. The potassium salt of compound 15 showed a high solubility and a good oral bioavailability (58%). Oral administration of the potassium salt remarkably improved the plasma glucose levels of female Zucker diabetic fatty rats at 1 mg/kg. Moreover, it did not cause a plasma volume increase or a cardiac enlargement in Wistar-Imamichi rats, even at 100 mg/kg.

In vivo antisense activity of ENA oligonucleotides targeting PTP1B mRNA in comparison of that of 2'-MOE-modified oligonucleotides.

The 2'-0-(2-methoxy)ethyl (2'-MOE)-modified gapmer antisense oligonucleotide ISIS113715, which targets protein-tyrosine phosphatase IB (PTP1B) mRNA, increases insulin sensitivity and normalizes plasma glucose levels in diabetic ob/ob and db/db mice. In the present study, the efficacy of the isosequential 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified oligonucleotide ENA-1 was compared with that of ISIS113715 in order to further improve the down-regulation of PTP1B in db/db mice. Intraperitoneal administration of ENA-1 more effectively decreased the plasma glucose levels in db/db mice than ISIS113715. Moreover, ENA-1 decreased the expression of PTP1B in the liver and fat of db/db mice more effectively than ISIS113715. These data indicate that ENA modifications enhance the ability of antisense oligonucleotides and make them superior to second-generation 2'-MOE modifications. We would like to thank to Drs. Shinya Tsutsumi and Kenji Kawai for the T(m) measurement and autoradiography experiments. ENA is a registered trademark of Mitsubishi-Kagaku Foods Corporation.

Identification of molecular target of AMP-activated protein kinase activator by affinity purification and mass spectrometry.

We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compound-immobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase (AMPK) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex I), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified.