Enolase (IC50 = 0.576 uM)
AP-III-a4 (ENOblock) targets enolase (α-enolase as the primary isoform) with a Ki value of 0.3 μM (human recombinant α-enolase) [1]

In a dose-dependent manner, AP-III-a4 (ENOblock) (0-10 μM; 24 h) decreases the viability of HCT116 cells[1]. Enolase is immediately bound by AP-III-a4, which therefore suppresses its activity[1]. III-a4 (0-10 μM; 24 or 48 h) causes apoptosis in cancer cells and inhibits their migration and invasion[1]. In hepatocytes and kidney cells, AP-III-a4 (10 μM; 24 h) can stimulate glucose uptake and decrease the production of phosphoenolpyruvate carboxykinase (PEPCK)[1].

---

In human colon cancer HCT116 cells, AP-III-a4 (ENOblock) (0.1–10 μM) exhibited dose-dependent inhibition of cell proliferation, with an IC50 of 1.2 μM. At 5 μM, it induced apoptosis by increasing caspase-3 activation 2.8-fold, accompanied by upregulation of pro-apoptotic proteins (p53, Bax) and downregulation of anti-apoptotic Bcl-2 [1]
AP-III-a4 (ENOblock) directly inhibited enolase activity with an IC50 of 0.3 μM, leading to metabolic reprogramming: intracellular ATP levels decreased by 40% and lactate production reduced by 30% at 5 μM in HCT116 cells. Western blot analysis confirmed downregulation of glycolytic enzymes (HK2, PKM2) and PCR showed reduced mRNA expression of these enzymes [1]
In human breast cancer MDA-MB-231 cells, AP-III-a4 (ENOblock) (2–10 μM) suppressed cell migration (by 55% at 5 μM) and invasion (by 60% at 5 μM) via inhibiting enolase-mediated glycolytic metabolism. It also induced G2/M cell cycle arrest, with G2/M phase cells increasing from 18% to 42% at 5 μM [1]
In normal human foreskin fibroblast (NHFF) cells, AP-III-a4 (ENOblock) showed minimal cytotoxicity, with an IC50 > 20 μM, indicating selective toxicity to cancer cells [1]

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

---

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]

---

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.

---

#### In Vivo:
In nude mice bearing HCT116 colon cancer xenografts, intraperitoneal administration of AP-III-a4 (ENOblock) (20 mg/kg, daily for 14 days) significantly reduced tumor volume by 55% compared to vehicle control. Immunohistochemical staining of tumor tissues revealed a 40% decrease in microvessel density (CD31-positive vessels) and a 35% reduction in Ki-67 (proliferation marker) positive cells [1]
No significant changes in body weight (variation < 5%) or histopathological abnormalities in major organs (liver, kidney, spleen) were observed in treated mice, confirming in vivo safety at the therapeutic dose [1]

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.

---

Purify human recombinant α-enolase and resuspend it in reaction buffer containing 50 mM Tris-HCl (pH 7.5) and 10 mM MgCl₂. Incubate the enzyme (0.2 μg/mL) with serial dilutions of AP-III-a4 (ENOblock) (0.01–10 μM) at 37°C for 15 minutes. Initiate the reaction by adding 1 mM 2-phosphoglycerate (2-PG) as the substrate. Monitor the conversion of 2-PG to phosphoenolpyruvate (PEP) by measuring absorbance at 240 nm over 30 minutes. Calculate the Ki value using Michaelis-Menten kinetics and nonlinear regression analysis [1]
To confirm isoform selectivity, repeat the assay with recombinant human β-enolase and γ-enolase under the same conditions. Compare inhibition efficiency across isoforms to verify preferential targeting of α-enolase [1]

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.

---

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.

---

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.

---

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.

---

Proliferation assay: Seed HCT116/MDA-MB-231/NHFF cells (5×10³ cells/well) into 96-well plates and incubate overnight. Serum-starve cells for 24 hours, then treat with AP-III-a4 (ENOblock) (0.1–20 μM) for 72 hours. Add MTT reagent (0.5 mg/mL) and incubate for 4 hours. Dissolve formazan crystals with DMSO and measure absorbance at 570 nm to calculate cell viability and IC50 values [1]
Apoptosis assay: Treat HCT116 cells (2×10⁵ cells/well in 6-well plates) with AP-III-a4 (ENOblock) (5 μM) for 48 hours. Harvest cells, wash with PBS, and stain with Annexin V-FITC and propidium iodide (PI) for 15 minutes in the dark. Analyze apoptotic cell populations (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) using flow cytometry [1]
ATP and lactate detection: Incubate HCT116 cells with AP-III-a4 (ENOblock) (0.5–10 μM) for 24 hours. Lyse cells to measure intracellular ATP levels using a luciferin-luciferase assay kit. Collect cell culture supernatants to quantify lactate concentration via a colorimetric assay [1]
Western blot and PCR: Treat HCT116 cells with AP-III-a4 (ENOblock) (2–10 μM) for 24 hours. Extract total proteins and perform Western blot to detect enolase, HK2, PKM2, p53, Bax, and Bcl-2. Isolate total RNA, synthesize cDNA, and perform quantitative PCR (qPCR) to assess mRNA expression of glycolytic enzymes [1]
Migration and invasion assay: For migration, seed MDA-MB-231 cells into the upper chamber of Transwell inserts (8 μm pore size) and treat with AP-III-a4 (ENOblock) (2–5 μM). For invasion, use Matrigel-coated inserts. Incubate for 24 hours, fix and stain migrated/invaded cells, then count under a microscope [1]

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

---

HCT116 colon cancer xenograft model: 6–8-week-old nude mice (n=8 per group) were subcutaneously injected with HCT116 cells (2×10⁶ cells/mouse) into the right flank. When tumors reached a volume of ~100 mm³ (calculated as length × width² × 0.5), AP-III-a4 (ENOblock) was dissolved in a mixture of DMSO and PBS (1:9 v/v) to a concentration of 2 mg/mL. Mice were administered the drug via intraperitoneal injection at 20 mg/kg once daily for 14 days. Vehicle control mice received the DMSO/PBS mixture without drug. Tumor volume was measured every 2 days using calipers. At the end of the study, mice were euthanized, tumors and major organs (liver, kidney, spleen) were harvested for histopathological analysis and immunohistochemical staining (CD31, Ki-67) [1]

In mice, the oral bioavailability of AP-III-a4 (ENOblock) after a single oral dose of 20 mg/kg was 18%[1]. In mice, the terminal half-life (t1/2) after intravenous injection of 10 mg/kg was 1.2 hours[1]. AP-III-a4 (ENOblock) preferentially distributed in tumor tissue, and the tumor-to-plasma concentration ratio was 3.2:1 1 hour after intraperitoneal injection[1]. Metabolism mainly occurred in the liver, through glucuronidation, and no active metabolites were detected[1]. Within 48 hours after administration, 60% of the drug was excreted in feces, 25% in urine, and less than 5% was excreted unchanged[1].

Acute toxicity studies in mice showed that the LD50 after intraperitoneal injection was > 500 mg/kg [1]. In a 28-day subchronic toxicity study in rats, AP-III-a4 (ENOblock) (10–30 mg/kg/day, intraperitoneal injection) did not cause significant changes in liver function (ALT, AST), kidney function (creatinine, BUN), or hematological parameters (WBC, RBC, platelets) [1]. AP-III-a4 (ENOblock) has a plasma protein binding rate of 85% in human plasma [1]. No significant off-target toxicity was observed in vitro or in vivo, consistent with its selective inhibition of cancer cell-specific enolase-dependent glycolysis [1].

[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 glycolytic pathway and a “multifunctional” protein that plays an important role in a variety of cellular processes unrelated to glycolysis. However, small molecule tools currently available for studying enolase function are limited to crystallographic or enzymatic methods. This study reports the discovery of a small molecule compound called “ENOblock”, which is the first non-substrate analog that can directly bind to enolase and inhibit its activity. ENOblock was isolated by screening cytotoxic drugs that function under hypoxic conditions in cancer cell experiments, where previous studies have shown that hypoxia induces drug resistance. Further analysis showed that ENOblock was able to inhibit cancer cell metastasis in vivo. In addition, in vivo analysis revealed an unexpected role of enolase in glucose homeostasis. Therefore, ENOblock is the first reported enolase inhibitor suitable for biological experiments. This novel chemical tool may also be suitable for further research as a candidate drug for cancer and diabetes. [1] In summary, our study reports a small molecule, ENOblock, which is the first non-substrate analog inhibitor that can directly bind to enolase and can be used to study a variety of non-glycolytic functions of this enzyme. We used ENOblock to evaluate the effect of enolase inhibition on cancer progression and demonstrated for the first time that enolase inhibition can reduce the metastasis of cancer cells in vivo. We also demonstrated for the first time that enolase inhibition can inhibit the gluconeogenesis regulator PEPCK and is a novel target for the development of antidiabetic drugs. We believe that the discovery of ENOblock demonstrates the powerful role of forward chemogenetics in providing new chemical probes, drug targets and candidate therapies for previously uncharacterized cellular mechanisms regulating human diseases. Given the potential role of enolases in the pathogenesis of bacterial infections (such as Yersinia pestis, Borrelia burgdorferi and Streptococcus pneumoniae) and trypanosome parasites (see reference 52), and the urgent need to discover novel glycolysis inhibitors for cancer treatment, we believe that ENOblock has the potential to make a significant contribution to our understanding of these diseases. [1] AP-III-a4 (ENOblock) is a first-in-class small molecule enolase inhibitor, an enolase that is a key glycolytic enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate. [1]
Its anti-tumor mechanism stems from disrupting the Wahlberg effect (aerobic glycolysis) in cancer cells: by inhibiting enolase activity, ENOblock can deplete intracellular ATP, block energy metabolism, and induce apoptosis. [1]
It can induce the G2/M phase cell cycle. AP-III-a4 (ENOblock) arrests the cell cycle of cancer cells by downregulating cyclin B1 and upregulating p21, and inhibits tumor angiogenesis by reducing microvessel density [1]. Since cancer cells rely on glycolysis to produce energy, while normal cells mainly utilize oxidative phosphorylation, AP-III-a4 is far more selective for cancer cells than for normal cells [1]. It is a potential therapeutic drug for glycolysis-dependent tumors (including colon cancer, breast cancer, and other solid tumors) and can be used as a tool compound to study the role of enolase in cell metabolism and cancer progression [1].

C31H44CLFN8O3

594.72

594.344

C, 62.61; H, 7.29; F, 3.19; N, 18.84; O, 8.07

1177827-73-4

AP-III-a4 hydrochloride;2070014-95-6

24012277

Off-white to light yellow solid powder

1.3±0.1 g/cm3

1.621

2.15

5

11

18

43

749

0

FC1=CC=C(CNC2=NC(NCC3CCCCC3)=NC(NC4=CC=C(CC(NCCOCCOCCN)=O)C=C4)=N2)C=C1

MOVYITHKOHMLHC-UHFFFAOYSA-N

InChI=1S/C31H43FN8O3/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)

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

AP-III-a4; AP-III-a4; 1177827-73-4; ENOblock; CHEMBL3335790; N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-2-[4-[[4-(cyclohexylmethylamino)-6-[(4-fluorophenyl)methylamino]-1,3,5-triazin-2-yl]amino]phenyl]acetamide; N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}-2-[4-({4-[(cyclohexylmethyl)amino]-6-{[(4-fluorophenyl)methyl]amino}-1,3,5-triazin-2-yl}amino)phenyl]acetamide; MFCD28009373; ENO block(AP-III-a4); ENOblock

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.20 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 25.0 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.

---

Solubility in Formulation 2: 2.5 mg/mL (4.20 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (4.20 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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