BMS-986256 (Afimetoran)

Specific TLR7 and TLR8 dual inhibitor

Inhibition of TLR7 and TLR8 signaling by BMS-986256

BMS-986256 (Afimetoran) is an indole-based small molecule that functions as a dual and selective inhibitor of TLR7 and TLR8 [1, 2]. Its potency has been demonstrated in various in vitro assays and a mouse lupus model in vivo [1, 2]. BMS-986256 is currently under phase II clinical investigation in patients with systemic lupus erythematosus (SLE) [3, 4].

BMS-986256 efficiently inhibits human (h)TLR7, mouse (m)TLR7, and hTLR8, but not mTLR8, as assessed using InvivoGen's HEK-Blue™ reporter cell lines overexpressing hTLR7, mTLR7, hTLR8, and mTLR8 (see figures).

BMS-986256 exerts inhibitory actions on both NF-κB and IRF pathways downstream hTLR7 and hTLR8, as assessed with THP1-Dual™-derived cells (see figures).

Importantly, BMS-986256 blocks the activation of TLR7 and TLR8 with no effect on other endosomal TLRs, i.e. TLR3 and TLR9 (see figures).

 More details

Mode of action

The specific binding mechanism of BMS-986256 is not known yet. However, it is hypothesized to behave similarly to other TLR7/8 antagonists by maintaining the inactive conformation of TLR7 and TLR8 receptors [2, 5]. Future research is needed to elucidate the exact mode of action of BMS-986256.

Key features of BMS-986256

• Indole-based small molecule
• Dual and selective inhibitor of TLR7- and TLR8-mediated NF-κB and IRF pathways
• Potent inhibitor of human TLR7, human TLR8, and mouse TLR7
• No action on mouse TLR8 overexpressed in HEK-Blue™ cells
• InvitroFit™: each lot of BMS-986256 is highly pure (≥95%) and functionally tested.

References:

1. Bristol-Myers Squibb company. 04.01.2018. WO 2018/005586 A1. [1,2,4] Triazolo [1,5-A] Pyridinyl substituted indole compounds.
2. Zheng H. et al., 2023. Recent Advances on Small-Molecule Antagonists Targeting TLR7. Molecules 28 (2), pp.634. ff10.3390/molecules28020634ff. ffhal-04564284.
3. Kalliokas. et al., 2021. Targeting TLR Signaling Cascades in Systemic Lupus Erythematosus and Rheumatoid Arthritis: An Update. Biomedicines 12(1):138.
4. NCT04895696. www.clinicaltrials.gov. As accessed in October 2024.
5. Vlach J. et al., 2020. Discovery of M5049: a novel selective Toll-Like Receptor 7/8 inhibitor for treatment of autoimmunity. J Pharmacol Exp Ther. 376:397.

Figures

BMS-986256 is a potent inhibitor of human TLR7, mouse TLR7, and human TLR8, but not mouse TLR8 signaling pathways in HEK cells overexpressing TLR7 or TLR8. HEK‑Blue™ cells overexpressing hTLR7 (A), mTLR7 (B), hTLR8 (C), or mTLR8 (D), were cultured with increasing concentrations of BMS-986256. After 3 hours of incubation, the following ligands were added: 100 ng/ml (A) or 300 ng/ml (B) R848, a TLR7/8 agonist, or 300 ng/ml (C) or 1 µg/ml (D) TL8-506, a TLR8 agonist. After overnight incubation, the neutralizing activity of BMS-986256 was determined by measuring the reduction of SEAP production in the supernatant using the QUANTI‑Blue™ detection reagent. Data are shown as a percentage (%) of maximal TLR activation with each agonist (without inhibitor; mean +SEM).

BMS-986256 is a potent inhibitor of NF-κB and IRF signaling pathways downstream of human TLR7 and TLR8. THP1-Dual™ hTLR7 KO-TLR8 (A) and THP1-Dual™ hTLR8 (B) cells were cultured in the presence of increasing concentrations of BMS-986256. After 3 hours of incubation, the following ligands were added: 1 µg/ml R848 (TLR7/8 agonist) (A), or 1 µg/ml TL8-506 (TLR8 agonist) (B). After overnight incubation, the neutralizing activity of BMS-986256 was determined by measuring the reduction of SEAP and Lucia luciferase production in the supernatant using the QUANTI-Blue™ and QUANTI-Luc™ 4 detection reagents, respectively. Data are shown as a percentage (%) of maximal TLR activation with each agonist (without inhibitor; mean ±SEM).

Abbreviations: hTLR7 = HEK-Blue™ hTLR7, mTLR7 = HEK-Blue™ mTLR7, hTLR8 = HEK-Blue™ hTLR8, mTLR8 = HEK-Blue™ mTLR8, hTLR3 = HEK-Blue™ hTLR3, hTLR9 = HEK-Blue™ hTLR9 cells.

BMS-986256 is a specific dual inhibitor of TLR7 and TLR8. HEK‑Blue™ cells overexpressing hTLR7, mTLR7, hTLR8, mTLR8, hTLR3, or hTLR9 were incubated with BMS-986256 (1 μM). After 3 hours of incubation, the following ligands were added: R848 30 ng/ml (hTLR7) or 300 ng/ml (mTLR7), TL8-506 100 ng/ml (hTLR8) or 3 µg/ml (mTLR8), Poly(I:C) HMW 50 ng/ml (hTLR3), and ODN 2006 500 ng/ml (hTLR9). After overnight incubation, the neutralizing activity of BMS-986256 was determined by measuring the reduction of SEAP production in the supernatant using QUANTI‑Blue™ detection reagent. Data are shown as optical density (OD) at 630 nm (mean +SEM).

Specifications

CAS number: 2171019-55-7

Formula: C₂₆H₃₂N₆O 

Molecular weight: 444.57 g/mol

Solubility: 11.25 mM (5 mg/ml) in DMSO

Working concentration: 

• in vitro: 2 nM - 1 μM for cellular assays, depending on cell type and agonist (see figures).
• in vivo: 0.25 mg/kg - 2.5 mg/kg, oral gavage, once daily, in NZB/W mouse model of lupus [1].

Quality control:

• Purity: ≥95% (UHPLC)
• The inhibition of human TLR7 by BMS-986256 is confirmed using HEK-Blue™ hTLR7 cells.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ hTLR2 and HEK-Blue™ hTLR4 cells.

Reference:

1. Bristol-Myers Squibb company. 04.01.2018. WO 2018/005586 A1. [1,2,4] Triazolo [1,5-A] Pyridinyl substituted indole compounds.

Contents

• 2 mg of lyophilized BMS-986256

 BMS-986256 is shipped at room temperature.

 Upon receipt, store BMS-986256 at -20 °C. Upon resuspension of  BMS-986256 in DMSO, prepare aliquots and store them at -20 °C.

 The resuspended product is stable for 1 month when properly stored.

 Avoid repeated freeze-thaw cycles.

Details

TLR7 and TLR8 are both activated by single-stranded (ss)RNA and RNA molecules found in immune complexes with RNA protein-binding autoantibodies [1]. Thus, these two TLRs play a beneficial role in clearing microbial infections, but they can also contribute to the pathogenesis of autoimmune diseases such as cutaneous and systemic lupus erythematosus (CLE/SLE) [1-3]. In addition, it is postulated that TLR7/8 may play a role in the cytokine storm in severe coronavirus disease 2019 (COVID-19) pneumonia [2, 3].

BMS986256 is described as an equipotent dual TLR7/TLR8 inhibitor using in vitro cellular assays [4]. However, in-house data suggest that the in vitro potency for each inhibitor depends on the choice of TLR7/TLR8-expressing cell lines and the type of agonists.

BMS986256 is currently under investigation as an oral treatment for SLE [5]. Interestingly, BMS-986256 demonstrates steroid-sparing effects in a mouse lupus model, a key feature that could help overcome glucocorticoid resistance and side effects in SLE patients [6].

Chemical structure of BMS-986256

References:

1. Vlach J. et al., 2020. Discovery of M5049: a novel selective Toll-Like Receptor 7/8 inhibitor for treatment of autoimmunity. J Pharmacol Exp Ther. 376:397.
2. Port A. et al., 2021. Phase 1 study in healthy participants of the safety, pharmacokinectics, and pharmacodynamics of enpatoran (M5049), a dual antagonist of toll-like receptors 7 and 8. Pharmacol Res Perspect 9(5):e00842.
3. Klopp-Schulze I. et al. 2022. Applying Modeling and Simulations for Rational Dose Selection of Novel Toll-Like Receptor 7/8 Inhibitor Enpatoran for Indications of High Medical Need. Clin Pharmacol Ther, 112: 297-306.
4. Bristol-Myers Squibb company. 04.01.2018. WO 2018/005586 A1. [1,2,4] Triazolo [1,5-A] Pyridinyl substituted indole compounds
5. NCT04895696. www.clinicaltrials.gov. As accessed in October 2024.
6. Dudhgaonkar S, et al., 2023. AB0132 Afimetoran (BMS-986256), an equipotent TLR7/8 antagonist, demonstrates steroid-sparing effects in a lupus mouse model. Annals of the Rheumatic Diseases 82:1245-1246.