EPAC2 ( IC50 = 0.43 μM )
EPAC2 (Exchange protein directly activated by cAMP 2) antagonist (IC50: 0.43 ± 0.05 μM for EPAC2 GEF activity inhibition). [1]

ESI-05 (0.01 μM-1 nM) inhibits cAMP-mediated EPAC2 GEF activity with an IC50 of 0.43 μM, but is completely blank in inhibiting EPAC1 GEF activity [1]. ESI-05 (1, 5, 10, and 25 μM; 5 Western Blot Analysis[1] Cell Line: HEK293 cells Concentration: 1, 5, 10, and 25 μM Incubation Time: 5 min Results: Results in dose-dependent manner. EPAC Selection A cAMP analog (007-AM) induces a reduction in Rap1 activation.

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ESI-05 inhibited cAMP-mediated EPAC2 guanine nucleotide exchange factor (GEF) activity in a biochemical assay with an apparent IC50 of 0.43 ± 0.05 μM. [1]
In a competition binding assay using 8-NBD-cAMP, ESI-05 competed with the fluorescent cAMP analog for binding to purified EPAC2 protein with an apparent IC50 of 0.4 ± 0.1 μM, representing approximately a 100-fold increase in affinity compared to cAMP (IC50 ~40 μM). [1]
ESI-05 (25 μM) did not significantly alter the activity of type I or type II protein kinase A (PKA) holoenzymes in the presence or absence of 100 μM cAMP, whereas the selective PKA inhibitor H89 completely blocked PKA activity. [1]
In HEK293 cells stably expressing full-length EPAC2, pretreatment with ESI-05 (1, 5, 10, 25 μM) led to a dose-dependent reduction of Rap1 activation induced by the EPAC-selective cAMP analog 007-AM. ESI-05 (25 μM) alone had no effect on basal Rap1 activation. [1]
In HEK293 cells stably expressing an EPAC2-based FRET sensor (EPAC2-FL), ESI-05 (10 μM) completely blocked the decrease in FRET (indicative of EPAC2 activation) induced by 3 μM 007-AM. [1]
ESI-05 (10 μM) also blocked the activation of the EPAC2-FL FRET sensor induced by the cAMP-elevating agents forskolin and isobutylmethylxanthine in live-cell imaging experiments. [1]
ESI-05 (10 μM) did not block the activation of an EPAC1-based FRET sensor (EPAC1-FL or EPAC1-camps) in response to 007-AM or forskolin/isobutylmethylxanthine. [1]
ESI-05 did not inhibit the activation of a PKA-based FRET sensor (AKAR3) in response to 8-Br-cAMP. [1]
Deuterium exchange mass spectrometry (DXMS) analysis suggested that the structurally related compound ESI-07 (and by inference, ESI-05) binds to the interface between the two cAMP-binding domains (CBDs) of EPAC2, stabilizing its autoinhibitory conformation. This binding site is not present in EPAC1, which contains only one CBD, explaining the isoform specificity. [1]

ESI-05 (2, 4 and 8 mg/kg) reduces neuronal cells in vivo by inhibiting p38/BIM [2]. Animal model: Intracranial hemorrhage (ICH) model [2] Dosage: 2, 4, 8 mg/kg Administration: Results: Decreased apoptosis rate of cortical nerve cells, phosphorylated p38, Bcl-2like protein 11 (BIM) and caspase- 3 Protein expression decreased accordingly.

Primary High-Throughput Screening (HTS) Assay: A fluorescence-based assay was developed where binding of the fluorescent cAMP analog 8-NBD-cAMP to purified full-length EPAC2 protein causes a dramatic increase in fluorescence. This assay, performed in a 384-well plate format, was used to screen a chemical library for compounds that could compete with 8-NBD-cAMP binding and inhibit EPAC2 activity. [1]
Secondary Biochemical GEF Activity Assay: The guanine nucleotide exchange factor (GEF) activity of purified, recombinant full-length EPAC1 or EPAC2 protein was measured. The assay monitors the decrease in fluorescence of Rap1 protein pre-loaded with a fluorescent GDP analog (MantGDP) as it is exchanged for unlabeled GDP in the presence of cAMP and the test compound. Reaction rates were calculated by fitting kinetic traces to a single exponential decay to determine inhibitory potency (IC50). [1]
Competition Binding Assay: The ability of compounds to compete with 8-NBD-cAMP for binding to purified EPAC2 was assessed. Various concentrations of the test compound or unlabeled cAMP were added to a fixed concentration mixture of EPAC2 and 8-NBD-cAMP. The dose-dependent decrease in 8-NBD-cAMP fluorescence was monitored to determine the apparent IC50 for competition. [1]
PKA Counter-Screening Assay: The kinase activities of reconstituted type I and type II PKA holoenzymes were measured spectrophotometrically using a coupled enzyme assay. A synthetic peptide substrate (Kemptide) was used. The assay was performed in a 96-well plate format to determine if test compounds affected PKA activity in the presence or absence of cAMP. [1]

Cell Line: HEK293 cells
Concentration: 1, 5, 10, and 25 μM
Incubation Time: 5 min
Result: Led to a dose-dependent reduction of the EPAC-selective cAMP analog (007-AM) induced Rap1 activation.

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Cellular Rap1 Activation Assay (Pull-down): HEK293 cells stably expressing full-length human EPAC1 or mouse EPAC2 were serum-starved, pretreated with ESI-05 or vehicle, and then stimulated with the EPAC-selective agonist 007-AM. Cell lysates were subjected to a pull-down assay using Ral-GDS-RBD-GST affinity beads to isolate GTP-bound Rap1 (Rap1GTP). The levels of Rap1GTP and total cellular Rap1 were detected by immunoblotting with a Rap1-specific antibody. [1]
FRET-Based Cellular Activation Assay: HEK293 cells stably expressing genetically encoded FRET biosensors (EPAC1-FL, EPAC2-FL, EPAC1-camps, or AKAR3) were used. For EPAC sensors (EPAC1/2-FL, EPAC1-camps), activation by agonists (e.g., 007-AM, forskolin/isobutylmethylxanthine) causes a decrease in FRET, measured as an increase in the CFP/YFP emission ratio (485/535 nm). For the PKA sensor (AKAR3), activation causes an increase in FRET, measured as a decrease in the emission ratio. Cells were plated in 96-well plates, and FRET changes were monitored in real-time using a microplate reader or live-cell imaging. Pretreatment with ESI-05 was used to assess its ability to block agonist-induced sensor activation. [1]

Intracranial hemorrhage (ICH) model
2, 4, and 8 mg/kg

[1]. Isoform-specific antagonists of exchange proteins directly activated by cAMP. Proc Natl Acad Sci U S A. 2012 Nov 6;109(45):18613-8.

[2]. Inhibition of EPAC2 Attenuates Intracerebral Hemorrhage-Induced Secondary Brain Injury via the p38/BIM/Caspase-3 Pathway. J Mol Neurosci. 2019 Mar;67(3):353-363.

ESI-05 is a noncyclic nucleotide small molecule that has been identified as an EPAC2-specific antagonist. [1]
It was discovered through high-throughput screening of a drug-like compound library using a fluorescence-based EPAC2 targeting detection method. [1]
Its mechanism of action involves binding to allosteric sites at the interfaces of two cAMP-binding domains (CBD-A and CBD-B) specific to the EPAC2 subtype, thereby locking the protein into its inactive, self-inhibitory conformation. This explains its specificity for EPAC2 rather than EPAC1. [1]
ESI-05 (and ESI-07) are among the first reported EPAC protein subtype-specific pharmacological probes, providing valuable tools for elucidating the different biological functions of EPAC1 and EPAC2. [1]

C16H18O2S

274.37792

274.103

C, 70.04; H, 6.61; O, 11.66; S, 11.68

5184-64-5

272513

White to off-white solid powder

1.129g/cm3

426.7ºC at 760mmHg

250ºC

1.562

4.833

0

2

2

19

376

0

CC1=CC=C(C=C1)S(=O)(=O)C2=C(C=C(C=C2C)C)C

CGPHOZWFSFNOEQ-UHFFFAOYSA-N

InChI=1S/C16H18O2S/c1-11-5-7-15(8-6-11)19(17,18)16-13(3)9-12(2)10-14(16)4/h5-10H,1-4H3

1,3,5-trimethyl-2-(4-methylphenyl)sulfonylbenzene

ESI-05; NSC-116966; ESI05; NSC116966; ESI 05; NSC 116966

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: 50~55 mg/mL (182.2~200.5 mM)
Ethanol: ~55 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.11 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.

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