Dissecting the Role of the Hydroxyl Moiety at C14 in (+)-Opioid-Based TLR4 Antagonists via Wet-Lab Experiments and Molecular Dynamics Simulations

Toll-like receptor 4 (TLR4) is pivotal as an innate immune
receptor,
playing a critical role in mediating neuropathic pain and drug addiction
through its regulation of the neuroinflammatory response. The nonclassical
(+)-opioid isomers represent a unique subset of TLR4 antagonists known
for their effective blood–brain barrier permeability. Despite
growing interest in the structure–activity relationship of
these (+)-opioid-based TLR4 antagonists, the specific impact of heteroatoms
on their TLR4 antagonistic activities has not been fully explored.
This study investigated the influence of the hydroxyl group at C14
in six (+)-opioid TLR4 antagonists (1–6) using wet-lab experiments and in silico simulations.
The corresponding C14-deoxy derivatives (7–12) were synthesized, and upon comparison with their corresponding
counterparts (1–6), it was discovered
that their TLR4 antagonistic activities were significantly diminished.
Molecular dynamics simulations showed that the (+)-opioid TLR4 antagonists
(1–6) possessed more negative binding
free energies to the TLR4 coreceptor MD2, which was responsible for
ligand recognition. This was primarily attributed to the formation
of a hydrogen bond between the hydroxyl group at the C-14 position
of the antagonists (1–6) and the
R90 residue of MD2 during the binding process. Such an interaction
facilitated the entry and subsequent binding of these molecules within
the MD2 cavity. In contrast, the C14-deoxy derivatives (7–12), lacking the hydroxyl group at the C-14
position, missed this crucial hydrogen bond interaction with the R90
residue of MD2, leading to their egression from the MD2 cavity during
simulations. This study underscores the significant role of the C14
hydroxyl moiety in enhancing the effectiveness of (+)-opioid TLR4
antagonists, which provides insightful guidance for designing future
(+)-isomer opioid-derived TLR4 antagonists.