Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling

Abstract

Deregulation of innate immune TLR4 signaling contributes to various diseases including neuropathic pain and drug addiction. Naltrexone is one of the rare TLR4 antagonists with good blood-brain barrier permeability and showing no stereoselectivity for TLR4. By linking 2 naltrexone units through a rigid pyrrole spacer, the bivalent ligand norbinaltorphimine was formed. Interestingly, (+)-norbinaltorphimine [(+)-1] showed ∼25 times better TLR4 antagonist activity than naltrexone in microglial BV-2 cell line, whereas (-)-norbinaltorphimine [(-)-1] lost TLR4 activity. The enantioselectivity of norbinaltorphimine was further confirmed in primary microglia, astrocytes, and macrophages. The activities of meso isomer of norbinaltorphimine and the molecular dynamic simulation results demonstrate that the stereochemistry of (+)-1 is derived from the (+)-naltrexone pharmacophore. Moreover, (+)-1 significantly increased and prolonged morphine analgesia in vivo. The efficacy of (+)-1 is long lasting. This is the first report showing enantioselective modulation of the innate immune TLR signaling.-Zhang, X., Peng, Y., Grace, P. M., Metcalf, M. D., Kwilasz, A. J., Wang, Y., Zhang, T., Wu, S., Selfridge, B. R., Portoghese, P. S., Rice, K. C., Watkins, L. R., Hutchinson, M. R., Wang, X. Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Keywords: MD-2; TLR4; enantioselective modulation; morphine analgesia; norbinaltorphimine.

Figure 1

Structures of naltrexone and its binary derivatives, (+)-1, (−)-1, and the meso isomer 2.

Figure 2

Norbinaltorphimine enantioselectively inhibits TLR4 signaling in the microglial BV-2 cell line. (+)-1 inhibited LPS-induced NO (A), TNF-α (B), and IL-1β (C) overproduction in BV-2 cell line, whereas (−)-1 showed no apparent inhibition toward LPS-induced NO (D), TNF-α (E), and IL-1β (F) overproduction in BV-2 cells.

Figure 3

Norbinaltorphimine enantioselectively inhibits TLR4 signaling in the primary microglial cells and primary astrocytes. (+)-1 inhibited LPS-induced NO (A, C) and TNF-α (B, D) overproduction in primary microglia (A, B) and primary astrocytes (C, D), whereas (−)-1 showed no apparent effect.

Figure 4

The calculated binding mode of norbinaltorphimine/MD-2 complex. A) View of the overall binding structure of norbinaltorphimine/MD-2 with the lowest energy during molecular dynamics simulations. B–D) Enlarged view of the interactions of (−)-1 (B), (+)-1 (C), and 2 (D) binding with MD-2. Ligands are shown as a ball-and-stick model, and MD-2 is shown as a cyan cartoon. Key residues of MD-2 in interacting with small-molecule antagonists are shown as green sticks. The yellow dash line represents a hydrogen bond.

Figure 5

The calculated binding process of norbinaltorphimine with MD-2. A) The free-energy profiles for (−)-1 (black), (+)-1 (blue), and 2 (red) binding with MD-2. The reaction coordinate was defined as the distance between the center of mass of the nonhydrogen atoms of ligands and that of the C-α atoms of the stable part of the so-called β-cup fold of MD-2. B–D) The binding snapshot for (−)-1 (B), (+)-1 (C), and 2 (D) at the entrance of MD-2 (cavity A). Key residues of MD-2 in recognizing norbinaltorphimine are shown as green sticks. Ligands are shown as a ball-and-stick model, and MD-2 is shown as a cyan cartoon.

Figure 6

The in vivo efficacy of (+)-1 on potentiating morphine analgesia. A, B) Intrathecal coadministration of morphine (15 μg) with (+)-1 (60 μg) produced a significant potentiation of morphine tail flick analgesia (A), as measured by area under the curve (B). C, D) (+)-1 even potentiated a second dose of morphine 24 h after (+)-1 administration (C) as measured by area under the curve (D); n = 5–6/group. **P < 0.01, ***P < 0.001.