A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation
Fig 4
Ugi compounds preferentially activate TLR4-HEK transfectants expressing human MD2.
A-B) HEK293 cells transfected with human TLR4/MD2 or mouse TLR4/MD2 (InVivogen) were stimulated with TLR4 agonists including MPL, PHAD, and Ugi compounds at 1 μg/ml to 4 ng/ml and with LPS at 25 ng/ml to 64 pg/ml for 24 h. SNs were analyzed for SEAP content by the QUANTI-Blue assay (InVivogen) and analysis at 650 nm by spectrophotometer. AUC (area under the curve) values were calculated as in Methods. No AUC value was calculated for LPS since it was utilized with a different dose range. C) HEK transfectants with cis- or trans-species expression of TLR4, MD2, and CD14 were stimulated with Ugi compounds in a dose response range of 9 μM to 1 nM or with LPS (boxes) at 75 ng/ml to 5 pg/ml for 48 h. Data was obtained as Vmax calculated over the initial linear portion of the kinetic absorbance measurements. The AUC (area under the curve) value for the dose response curve of Vmax vs log10 (molar units) was calculated for each compound using Trapezoidal rule.