A2B Adenosine Receptor (A2B AR) - Positive Allosteric Modulator (PAM) [2]

Runx2 and Osterix mRNA expression can be significantly increased by KI-7 (1 μM; 5–21 days; mesenchymal stem cells) [1]. Cell viability is significantly increased at both differentiation stages by KI-7 (1 μM; 15–21 days) [1]. It has been demonstrated that KI-7, a positive allosteric modulator of the A2B adenosine receptor in MSCs, enhances the in vitro osteoblast differentiation-promoting actions of both adenosine and synthetic orthosteric A2B adenosine receptor agonists. KI-7, BAY 60-6583, and NECA all significantly increased the production of IL-6. Even though this effect only becomes noticeable at day 21, KI-7 amplifies the effects of orthotopic agonists at both differentiation stages [1].

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In human mesenchymal stem cells (MSCs) undergoing osteogenic differentiation, KI-7 (1 μM) alone did not significantly affect basal intracellular cAMP levels, indicating it has no intrinsic agonist efficacy. [2]
- KI-7 potentiated cAMP accumulation evoked by the selective A2B AR agonist BAY 60-6583 (5 and 50 nM) in both undifferentiated and differentiated MSCs. This potentiation was completely blocked by the selective A2B AR antagonist MRS1706. [2]
- KI-7 altered the time course of cAMP production in response to A2B AR agonists. While agonist-mediated cAMP levels peaked at days 5-9 and then declined, in the presence of KI-7, cAMP levels remained elevated throughout the differentiation process (up to 21 days). [2]
- KI-7 exhibited probe-independent allosteric modulation, potentiating cAMP production stimulated by the non-selective agonists NECA and adenosine at all stages of MSC differentiation. These effects were also blocked by MRS1706. [2]
- In MSC cultures, KI-7 (1 μM) alone significantly increased the mRNA expression of osteoblast-related transcription factors Runx2 and Osterix at various differentiation time points (days 5, 15, 21). It also potentiated the increases in Runx2 and Osterix expression induced by NECA (100 nM) and BAY 60-6583 (5 nM). [2]
- KI-7 (1 μM) alone increased the expression of the osteoblast marker proteins alkaline phosphatase (ALP) and osteocalcin. Furthermore, it potentiated the effects of NECA and BAY 60-6583 on the expression of these markers. [2]
- KI-7 (500 nM - 5 μM) alone induced a concentration-dependent increase in osteoblast mineralization, as measured by OsteoImage™ staining. This effect was completely abrogated by the A2B AR antagonist MRS1706 and by adenosine deaminase (ADA), which removes endogenous adenosine, suggesting KI-7 potentiates the effects of endogenous adenosine. [2]
- KI-7 (500 nM - 5 μM) potentiated osteoblast mineralization evoked by both NECA (100 nM) and BAY 60-6583 (5 nM) in a concentration-dependent manner at all differentiation stages (days 9, 15, 21). These effects were blocked by MRS1706. [2]
- In the early phase of differentiation (days 5-9), treatment with KI-7 (1 μM) alone or in combination with NECA/BAY 60-6583 reduced IL-6 release, favoring the spontaneous decrease in IL-6 production associated with differentiation. [2]
- In the late phase of differentiation (days 15-21), KI-7 (1 μM) alone or in combination with NECA/BAY 60-6583 caused a sustained increase in IL-6 release. [2]
- At days 15 and 21 of differentiation, KI-7 (1 μM) alone or in combination with NECA (100 nM) significantly increased the viability of differentiated osteoblasts, as measured by MTS assay. This pro-survival effect was blocked by MRS1706. [2]

cAMP Accumulation Assay: MSCs were plated in 24-well plates and differentiated for various times. Cells were pre-incubated with medium containing the phosphodiesterase inhibitor Ro 20-1724 (20 μM) and adenosine deaminase (ADA, 1 U/mL). Cells were then treated for 15 min with A2B AR agonists (BAY 60-6583, NECA, or adenosine) and/or the allosteric modulator KI-7 (1 μM). The specificity was tested using the A2B AR antagonist MRS1706 (15 nM). Intracellular cAMP levels were measured using a competitive protein binding method. [2]
- Gene Expression Analysis (RT-qPCR): MSCs were cultured and treated every two days with osteogenic medium alone (control), BAY 60-6583 (5 nM), NECA (100 nM), or KI-7 (1 μM), alone or in combination. After 5, 15, or 21 days, total RNA was extracted. Reverse transcription was performed, and the resulting cDNA was used for qPCR with primers specific for Runx2, Osterix, ALP, and osteocalcin. mRNA levels were normalized against β-actin. [2]
- Mineralization Assay (OsteoImage™): MSCs were seeded and treated every two days with osteogenic medium containing the test compounds (KI-7 500 nM - 5 μM, NECA 100 nM, BAY 60-6583 5 nM, alone or in combination) for 9, 15, or 21 days. Cells were also treated with forskolin (1 μM) and 8-Br-cAMP (100 nM - 10 μM). Mineralization was quantified using OsteoImage™ Staining Reagent, which binds to the hydroxyapatite portion of bone-like nodules. Fluorescence was measured at excitation/emission wavelengths of 485/535 nm. [2]
- Mineralization Assay (Alizarin Red S): For qualitative analysis, MSCs differentiated for 21 days under various treatments were fixed, washed, and stained with 2% alizarin red S solution for 5 minutes. After washing and air-drying, images of the stained mineral nodules were captured using a light microscope. [2]
- Cell Viability Assay (MTS): MSCs were seeded in 96-well plates and cultured in osteogenic medium for 21 days in the presence of NECA (100 nM), KI-7 (1 μM), and/or MRS1706 (15 nM). At days 15 and 21, cell viability was assessed by adding MTS reagent following the manufacturer's instructions and measuring the absorbance. [2]
- IL-6 Production Assay (ELISA): MSCs were cultured in osteogenic medium for 5-21 days with NECA (100 nM), BAY 60-6583 (5 nM), and/or KI-7 (1 μM), with or without MRS1706. At each time point, the conditioned medium was collected, and the levels of secreted IL-6 were quantified using an ELISA kit. [2]

[1]. Allosteric modulators of human A2B adenosine receptor. Biochim Biophys Acta. 2014;1840(3):1194-1203.

[2]. Osteoblast differentiation and survival: A role for A2B adenosine receptor allosteric modulators. Biochim Biophys Acta. 2014;1843(12):2957-2966.

KI-7 (2-(1-benzyl-1H-indol-3-yl)-2-oxo-N-phenylacetamide) is a 3-keto-indole derivative identified as a first-in-class positive allosteric modulator of the A2B adenosine receptor. Previous work characterized it as having high efficacy and selectivity for the A2B AR over other adenosine receptor subtypes (A1, A2A, A3). [2]
- The mechanism of action involves facilitating A2B AR-Gs protein coupling, thereby increasing the functional response of orthosteric agonists on the cAMP signaling pathway without directly activating the receptor itself. [2]
- In the context of bone biology, this study demonstrates that KI-7 promotes osteoblast differentiation from MSCs by potentiating the effects of both synthetic A2B AR agonists and endogenous adenosine. It enhances the expression of osteogenic markers, accelerates mineralization, and improves the survival of mature osteoblasts. [2]
- The compound modulates IL-6 release in a biphasic, differentiation stage-dependent manner: it reduces IL-6 in early stages to favor differentiation and increases IL-6 in late stages to promote osteoblast survival. This suggests that positive allosteric modulation of A2B AR by KI-7 could be a promising therapeutic approach for bone diseases like osteoporosis by simultaneously promoting bone formation and ensuring the survival of bone-forming cells. [2]

C23H18N2O2

354.401225566864

354.136

1489263-00-4

73350966

White to off-white solid powder

4.3

1

2

5

27

523

0

O=C(C(NC1C=CC=CC=1)=O)C1=CN(CC2C=CC=CC=2)C2C=CC=CC=21

MQMGZFRIEZRHHJ-UHFFFAOYSA-N

InChI=1S/C23H18N2O2/c26-22(23(27)24-18-11-5-2-6-12-18)20-16-25(15-17-9-3-1-4-10-17)21-14-8-7-13-19(20)21/h1-14,16H,15H2,(H,24,27)

2-(1-benzylindol-3-yl)-2-oxo-N-phenylacetamide

KI7 KI 7 KI-7

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 : ~250 mg/mL (~705.42 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.87 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.)