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Molecular Determinants of GS-9620-Dependent TLR7 Activation

Fig 2

Structure-based mutational analysis identifies residues in TLR7 that are essential for GS-9620 in vitro activity.

(a) Three dimensional molecular model of TLR7 endo-lysosomal domain (left) and magnified view of GS-9620 docked in TLR7 (right). (b,c) Fold increase in NF-κB-luciferase reporter activity upon GS-9620, resiquimod or DMSO control stimulation in Huh7 cells that were transfected with control vector, wild-type TLR7 or point mutants of TLR7. Four 2-fold dose titrations (left to right) were performed starting at 5μM for GS-9620, or 10 μM of resiquimod. Bar graphs show fold change of reporter activity relative to DMSO control, and error bars shown represent the mean of triplicate assay conditions and SEM. Representative data shown from three independent experiments with similar results. Area under the curve (AUC) calculations were performed to quantify reporter activity observed with titrated compound concentrations for each of the TLR7 variants. With GS-9620 stimulation the variants Y356S, F408A, D555A, L557D, and L557Y/T586G, and with R848 stimulation the variants Y356S, F408A, and D555A show a 4-fold or greater reduction in reporter activity compared to TLR7 WT, as assessed by AUC calculation. Therefore, these variants are viewed as significantly compromised in response to the respective compound.

Fig 2