IC50: 0.576 uM (enolase)[1]

In a dose-dependent manner, AP-III-a4 hydrochloride (ENOblock) (0-10 μM; 24 h) decreases the viability of HCT116 cells [1]. Enolase's activity is inhibited by AP-III-a4 hydrochloride, which binds to it directly [1]. AP-III-a4 hydrochloride (0–10 μM; 24 or 48 h) causes cancer cells to undergo apoptosis and inhibits their migration and invasion [1]. AP-III-a4 hydrochloride (10 μM; 24 h) suppresses the production of phosphoenolpyruvate carboxykinase (PEPCK) and promotes hepatocytes and renal cells to ingest glucose [1].

In zebrafish, AP-III-a4 hydrochloride (ENOblock) (10 μM; 96 h) suppresses the spread of cancer cells and the gluconeogenesis regulator PEPCK[1].

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The zebrafish (Danio rerio) cancer cell xenograft model is gaining increasing research prominence as a validated, convenient tool for testing candidate cancer drugs in vivo. In addition, zebrafish is a relevant vertebrate platform for predicting toxicological effects in mammals. We observed that 10 μM ENOblock treatment of developing zebrafish larvae was nontoxic (Figure 4, panels A–C). Employing a recently published zebrafish tumor xenograft model validated for anticancer drug testing, we observed that ENOblock treatment reduced cancer cell dissemination, suggesting an inhibition of cancer cell migration and invasion processes (Figure 4, panels D,E).[1]

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ENOblock Down-regulates PEPCK Expression and Induces Glucose Uptake in Vivo[1]
To investigate the effects of ENOblock on glucose homeostasis in vivo, we selected the zebrafish, because this animal model provides a convenient, rapid experimental format requiring small amounts of test compound. Moreover, it has been shown that zebrafish and mammals share similar glucose regulatory responses. Adult zebrafish treated with ENOblock or rosiglitazone showed down-regulated hepatic PEPCK expression (Figure 6, panels a,b), which confirmed our cell-based findings. The fluorescent glucose probe 2-NBDG has been used to assess glucose uptake in zebrafish larvae, which are transparent and allow visualization of 2-NBDG fluorescence (e.g., ref 26). We observed that ENOblock treatment induced glucose uptake in zebrafish larvae (Figure 6, panels c–e). As a comparison, we also tested the effect of emodin (6-methyl-1,3,8-trihydroxyanthraquinone, a biologically active plant constituent that is known to promote cellular glucose uptake). 2-NBDG fluorescent signal in lysed larvae was measured using a fluorescent plate reader (Figure 6, panel d). Results from this approach confirmed that ENOblock treatment induced glucose uptake in vivo. Fluorescence microscopy analysis of 2-NBDG treated larvae showed that emodin treatment increased glucose uptake (Figure 6, panel e). 2-NBDG uptake was quantified by measuring 2-NBDG fluorescence intensity in the zebrafish larvae eye at 72 hpf, because this tissue has been show to express a relatively large number of glucose transporter isoforms at this stage of development. Image J anaylsis confirmed that ENOblock or emodin treatment could promote glucose uptake in the zebrafish.

ENOblock Binds to Enolase and Inhibits Its Activity[1]
Affinity chromatography was used to identify the cellular target for AP-III-a4. Target identification strategies for the triazine library used in this study are relatively straightforward, because the molecules contain a built-in linker moiety. This allows conjugation to an affinity matrix with reduced risk of compromising biological activity. Silver staining of proteins eluted from the AP-III-a4 affinity matrix is shown in Figure 2, panel a. Mass spectrometry analysis revealed that two protein bands of approximately 45 kD mass were subunits of enolase, a glycolysis enzyme, and a protein band of approximately 40 kD was actin (Figure 2, panel b; the entire mass spectrometry analysis for AP-III-a4 is shown in Supplementary Figure 2 and Supplementary Table 1). However, AP-III-a4 did not affect actin polymerization (Supplementary Figure 3), indicating that actin is not an active target. Thus, we renamed molecule AP-III-a4 “ENOblock”. ENOblock binding to enolase in cancer cell lysates was confirmed by Western blot analysis of proteins eluted from the ENOblock affinity matrix. Competition analysis with free ENOblock inhibited enolase binding to the ENOblock affinity matrix (Figure 2, panel c). Moreover, ENOblock could bind to purified human enolase, suggesting a direct interaction between ENOblock and enolase (Figure 2, panel d). Subsequent analysis showed that enolase activity can be inhibited by ENOblock dose-dependently (Figure 2, panel e; as an additional control, we also tested another non-hit compound from the tagged triazine library, AP-I-f10 (3), which was shown to not reduce enolase activity (Figure 2, panel f)). Further biochemical analysis showed that the half maximal inhibitory concentration (IC50) of enolase inhibition by ENOblock is 0.576 μM (Supplementary Figure 4).The role of enolase in enhancing cancer cell survival under hypoxia was confirmed by siRNA-mediated knock-down of enolase expression (Supplementary Figure 5). To test that ENOblock treatment under hypoxia induced cytotoxicity, rather than inhibition of cell proliferation, cancer cells were stained with trypan blue (Supplementary Figure 6). ENOblock-treated cells showed increased trypan blue uptake under hypoxia, which confirmed the induction of cell death.

Cell Viability Assay[1]
Cell Types: HCT116
Tested Concentrations: 1.25, 2.5, 5 and 10 μM
Incubation Duration: 24 h
Experimental Results: Induced higher levels of HCT116 colon cancer cell death in hypoxic conditions compared to normoxia.

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Western Blot Analysis[1]
Cell Types: HCT116
Tested Concentrations: 1.25, 2.5, 5 and 10 μM
Incubation Duration: 24 h for AKT, 48 h for Bcl-Xl
Experimental Results: Bound to enolase in cell lysate and bound to purified enolase. diminished the expression of AKT and Bcl-Xl, which are negative regulators of apoptosis. Cell Invasion Assay[1]
Cell Types: HCT116
Tested Concentrations: 0.156, 0.312, 0.625, 1.25 and 2.5 μM
Incubation Duration: 24 h
Experimental Results: Dramatically inhibits cancer cell invasion at a treatment concentration of 0.625 μM.

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Cell Migration Assay [1]
Cell Types: HCT116
Tested Concentrations: 0.625, 1.25 and 2.5 μM
Incubation Duration: 24 h
Experimental Results: Inhibited cell migration dose-dependently.

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RT-PCR[1]
Cell Types: Huh7 and HEK
Tested Concentrations: 10 μM
Incubation Duration: 24 h
Experimental Results: Induced glucose uptake and inhibited PEPCK expression.

Animal/Disease Models: The zebrafish cancer cell HCT116 xenograft model[1]
Doses: 10 μM
Route of Administration: 96 h
Experimental Results: diminished cancer cell dissemination. Inhibited PEPCK expression and induced glucose uptake. Inhibited adipogenesis and foam cell formation.

[1]. A Unique Small Molecule Inhibitor of Enolase Clarifies Its Role in Fundamental Biological Processes.ACS Chem. Biol., 2013, 8 (6), pp 1271–1282.

Enolase is a component of the glycolysis pathway and a "moonlighting" protein, with important roles in diverse cellular processes that are not related to its function in glycolysis. However, small molecule tools to probe enolase function have been restricted to crystallography or enzymology. In this study, we report the discovery of the small molecule "ENOblock", which is the first, nonsubstrate analogue that directly binds to enolase and inhibits its activity. ENOblock was isolated by small molecule screening in a cancer cell assay to detect cytotoxic agents that function in hypoxic conditions, which has previously been shown to induce drug resistance. Further analysis revealed that ENOblock can inhibit cancer cell metastasis in vivo. Moreover, an unexpected role for enolase in glucose homeostasis was revealed by in vivo analysis. Thus, ENOblock is the first reported enolase inhibitor that is suitable for biological assays. This new chemical tool may also be suitable for further study as a cancer and diabetes drug candidate.[1]

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In summary, our study reports the small molecule ENOblock, which is the first nonsubstrate analogue inhibitor that directly binds to enolase and can be used to probe the various nonglycolytic functions of this enzyme. We have utilized ENOblock to assess the effect of enolase inhibition on cancer progression and show for the first time that enolase inhibition can reduce cancer cell metastasis in vivo. We also show for the first time that enolase inhibition can suppress the gluconeogenesis regulator PEPCK and is a new target for developing antidiabetic drugs. We believe that the discovery of ENOblock is a testament to the power of forward chemical genetics to provide new chemical probes, drug targets, and candidate therapeutics for previously uncharacterized cellular mechanisms regulating human disease. In light of the potential role of enolase in the pathogenesis of bacterial infections (such as Yersinia pestis, Borrelia spp., and Streptococcus pneumonia) and trypanosomatid parasites (reviewed in ref 52), in addition to the need to discover new glycolysis inhibitors for cancer therapy, we believe that ENOblock has the potential to make significant contributions to our understanding of these disorders.[1]

C31H44CLFN8O3

631.18

630.32

2070014-95-6

AP-III-a4;1177827-73-4

78357810

White to yellow solid powder

6

11

18

44

749

0

C1CCC(CNC2=NC(=NC(NC3=CC=C(C=C3)CC(NCCOCCOCCN)=O)=N2)NCC2C=CC(F)=CC=2)CC1.Cl

UYGRNXLHKIBHMM-UHFFFAOYSA-N

InChI=1S/C31H43FN8O3.ClH/c32-26-10-6-25(7-11-26)22-36-30-38-29(35-21-24-4-2-1-3-5-24)39-31(40-30)37-27-12-8-23(9-13-27)20-28(41)34-15-17-43-19-18-42-16-14-33;/h6-13,24H,1-5,14-22,33H2,(H,34,41)(H3,35,36,37,38,39,40);1H

N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-2-[4-[[4-(cyclohexylmethylamino)-6-[(4-fluorophenyl)methylamino]-1,3,5-triazin-2-yl]amino]phenyl]acetamide;hydrochloride

AP-III-a4 hydrochloride; AP-III-a4 (hydrochloride); 2070014-95-6; AP-III-a4 (ENOblock); N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-2-[4-[[4-(cyclohexylmethylamino)-6-[(4-fluorophenyl)methylamino]-1,3,5-triazin-2-yl]amino]phenyl]acetamide;hydrochloride; ENOblock Hcl; ENOblock hydrochloride; AP-III-a4(ENOblock) hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 53 mg/mL (83.97 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.30 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.30 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.30 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)