HIV protease inhibitor saquinavir inhibits toll-like receptor 4 activation by targeting receptor dimerization

Toll-like receptor 4 (TLR4) is crucial in induction of innate immune response through recognition of invading pathogens or endogenous alarming molecules. Ligand-triggered dimerization of TLR4 is essential for the activation of NF-κB and IRF3 through MyD88- or TRIF-dependent pathways. Saquinavir (SQV), an FDA-approved HIV protease inhibitor, has been shown to attenuate the activation of NF-κB induced by HMGB1 by blocking TLR4-MyD88 association in proteasome independent pathway. This study aims to define whether SQV is an HMGB1-specific and MyD88-dependent TLR4 signaling inhibitor and which precise signaling element of TLR4 is targeted by SQV.

PMA differentiated human THP-1 macrophages or reconstituted HEK293 cells were pretreated with SQV before stimulated by different TLR agonists. TNF-α level was evaluated through ELISA assay. NF-κB activation was analyzed using NF-κB SEAP reporting system. The levels of MyD88/TRIF pathways-related factors were examined by immunoblot. TLR4 endocytosis was assessed by immunocytochemistry. TLR4 dimerization was determined using immunoprecipitation between different tagged TLR4 and an in silico molecular docking experiment was performed to explore the possible binding site of SQV on its target.

Our data showed that SQV suppresses both MyD88- and TRIF-dependent pathways in response to lipopolysaccharide (LPS), a critical sepsis inducer and TLR4 agonist, leading to downregulation of NF-κB and IRF3. SQV did not suppress MyD88-dependent pathway triggered by TLR1/2 agonist Pam3csk4. In the only TRIF-dependent pathway, SQV did not alleviate IRF3 phosphorylation induced by TLR3 agonist Poly(I:C). Furthermore, dimerization of TLR4 following LPS or HMGB1 stimulation was decreased by SQV.

We concluded that TLR4 receptor complex is one of the mammalian targets of SQV, and TLR4-mediated immune responses and consequent risk for uncontrolled inflammation could be modulated by FDA-approved drug SQV.