TLR4 Targeting Probe Design Using in silico Structural Insights

TLR4 Targeting Probe Design Using in silico Structural Insights Andre A. Aguirre 1,2 , Sandun Perera 1,2 , Sidath C. Kumarapperuma 1,2 Affiliation: ¹Research Imaging Institute, 2 University of Texas Health Science Center at San Antonio Background and Purpose: Toll-like receptor 4 (TLR4) is a primary mediator of innate immune activation implicated in the early pathology of Alzheimer’s disease (AD).Aggregated amyloid-beta (Aβ) and hyperphosphorylated tau directly activate TLR4 15–20 years prior to clinical diagnosis. Noninvasive visualization via Positron Emission Tomography (PET) could enable early detection and therapeutic monitoring of these processes. Because current imaging probes lack the specificity to resolve this activation, there is a need for high-affinity tracers that target the TLR4-MD-2 complex. This study aimed to characterize the Lipopolysaccharide (LPS) binding pocket within the MD-2 co-receptor and identify key residues to guide the design of high-affinity, novel imaging probes and therapeutics. Methods: The human MD-2-LPS-TLR4 complex (PDB ID: 3FXI) was preparedIn Silico.Non-protein components were removed, and the heterodimer was isolated to generate an induced-fit, holo-apo conformation.Structural integrity was verified via clash-check to ensure biological relevance for Fpocket (v3.1) analysis.A custom 59-point color gradient quantified surface cavities based on druggability, volume, and depth.Pocket 6 was identified as the functionally relevant site and further analyzed for its physiochemical properties. Residues Phe 121 , Phe 126 , and Tyr 131 on MD-2 and Phe 440 and Phe 463 on TLR4 were investigated and modeled. Results: Computational analysis identified 59 potential surface cavities.Pocket 6, corresponding to the LPS-binding site, exhibited a Druggability Score of 0.984, binding volume of 941.353Å 3 , Hydrophobicity Score of 88.333, and a mean local hydrophobic density of 71.152.These metrics confirm a highly hydrophobic environment suitable for lipid-chain engagement.Structural modeling of critical residues identifiedPhe 121 and Tyr 131 as anchor sites, and Phe 126 or the Phe 440 /Phe 463 dimerization interface as potential modulatory targets. Conclusions: This study provides a structural and chemical map of the TLR4-MD-2 complex, identifying anchors for probe stabilization and residues with antagonistic potential.These findings support the rational design of high-affinity PET tracers and inhibitors to quantify or modulate TLR4 dynamics in vivo during pre-symptomatic AD, offering an early window for premature detection and therapeutic monitoring.