M5049 (Enpatoran)

Specific TLR7 and TLR8 dual inhibitor

Inhibition of TLR7 and TLR8 signaling by M5049

M5049 (also known as Enpatoran) is a small molecule that functions as a dual and selective inhibitor of TLR7 and TLR8 [1]. Its potency has been demonstrated in a range of in vitro assays and mouse lupus models in vivo [1]. M5049 efficiently inhibits human (h)TLR7, mouse (m)TLR7, and hTLR8, but not mTLR8, as assessed using InvivoGen's reporter cell lines HEK-Blue™ hTLR7, mTLR7, hTLR8, and mTLR8 (see Figure 1).
 

Mode of action:

Structural studies have revealed that M5049 binds and stabilizes the hTLR8 dimer in its resting (inactive) state, antagonizing the binding of any TLR8 ligands [1]. A similar mode of action has been suggested for TLR7 inhibition.
Importantly, M5049 blocks the activation of TLR7 and TLR8 without having any effect on other endosomal TLRs, i.e. TLR3 and TLR9 (see Figure 3).
 

Key features of M5049:

• M5049 specifically inhibits human TLR7, human TLR8, and mouse TLR7.
• M5049 acts as an antagonist that prevents the binding of TLR7 and TLR8 ligands to their receptors.
• InvitroFit™: each lot of M5049 is highly pure (≥95%) and functionally tested.

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. 

Figures

Figure 1: M5049 is a potent inhibitor of human TLR7, mouse TLR7, and human TLR8, but not mouse TLR8 signaling pathways. HEK‑Blue™ cells expressing hTLR7 (A), mTLR7 (B), hTLR8 (C), or mTLR8 (D), were cultured with increasing concentrations of M5049. After 3 hours of incubation, the following ligands were added: 30 ng/ml R848, a TLR7/8 agonist (A and B), or 30 ng/ml (C) or 300 ng/ml (D) TL8-506, a TLR8 agonist. After overnight incubation, the neutralizing activity of M5049 was determined by measuring the reduction of SEAP production in the supernatant using the HEK‑Blue™ detection reagent. Data are shown as a percentage (%) of maximal TLR activation with each agonist (without inhibitor; mean +SEM).

Figure 2: M5049 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 M5049. 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 M5049 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).

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.

Figure 3: M5049 is a specific dual inhibitor of TLR7 and TLR8. HEK‑Blue™ cells expressing hTLR7, mTLR7, hTLR8, mTLR8, hTLR3, hTLR4, or hTLR9 were incubated with M5049 (1 μM). After 3 hours of incubation, the following ligands were added: R848 30 ng/ml (hTLR7) or 100 ng/ml (mTLR7), TL8-506 30 ng/ml (hTLR8) or 300 ng/ml (mTLR8), Poly(I:C) HMW 50 ng/ml (hTLR3), LPS-EK 1 ng/ml (hTLR4), and ODN 2006 500 ng/ml (hTLR9). After overnight incubation, the neutralizing activity of M5049 was determined by measuring the reduction of SEAP production in the supernatant using the HEK‑Blue™ detection reagent. Data are shown as optical density (OD) at 630 nm (mean +SEM).

Specifications

CAS number: 2101938-42-3

Formula: C₁₆H₁₅F₃N₄ 

Molecular weight: 320.32 g/mol

Solubility: 62.4 mM (20 mg/ml) in H₂O

Working concentration:  40 nM - 1 μM for cellular assays

Quality control:

• Purity: ≥95% (UHPLC)
• The inhibition of human TLR7, human TLR8, and mouse TLR7 by M5049 has been confirmed using cellular assays.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ hTLR2 and HEK-Blue™ hTLR4 cells.

Contents

• 5 mg of lyophilized M5049

 M5049 is shipped at room temperature.

 Upon receipt, store M5049 at -20 °C. Upon resuspension of  M5049 in H₂O, prepare aliquots and store them at -20 °C.

 The resuspended product is stable for 6 months when properly stored.

 Avoid repeated freeze-thaw cycles.

Details

TLR7 and TLR8 are both activated by single-stranded (ss)RNA but also 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]. M5049 is currently under investigation as an oral treatment for lupus (NCT04647708) and severe COVID-19 (NCT04448756).

Chemical structure of M5049

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.