Preclinical Mechanistic Study On Developing Cathepsin V As A Novel Target For Tissue Injury Protection/Anti-Inflammation

Background:Extracellular disulfide HMGB1, via TLR4, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A high throughput screen identified first generation HIV-PI Saquinavir (SQV) as a potent inhibitor of HMGB1-TLR4driven cytokine production and murine liver ischemia/reperfusion model. We further reported on the identification of mammalian Cathepsin V, a protease, as a novel target of SQV. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between Cathepsin V with TLR4, indicating cathepsin V involves in TLR4signaling.Purposes and Methods:In this study, we expand these evidence for SQV and Cathepsin V. We utilize SQV and another known CTSV inhibitor SID26681509(SID) as chemical probes to reveal insights into the mechanism for CTSV participating TLR4signaling, as well as the nature of CTSV-TLR4interaction and the anti-inflammation property of SQV.Results:Here we report that, at receptor level, SQV specifically suppresses TLR4induced MyD88-IRAK4phosphorylation and TRIF-IRF3phosphorylation. While at NF-κB level, SQV efficiently attenuates TNF-a production or NF-κB activation triggered by diverse inflammatory stimuli including TLR1/2ligand Pam3CSK4, TLR4ligand LPS, TLR3ligand Poly (I:C) plus IL-1R ligand IL-1β or TNFR ligand TNF-a. And these properties are shared by the known CTSV inhibitor SID. Hinted from the mechanistic properties of SQV and SID266, we find that CTSV physically interacts with IRAK4in PMA-differentiated THP-1cells stimulated with HMGB1, which could be diminished by SQV administration. The genetic knockdown of CTSV diminishes TNF-α release triggered by MyD88-dependent Pam3CSK4and HMGB1but not TRIF-dependent Poly(I:C). When given at same concentration, SQV exerts stronger downregulation on TLR4-MyD88-IRAK1degradation than SID, a same trend as of the prevention on CTSV-TLR4interaction and an opposite one as of the inhibition on CTSV protease activity. Interestingly, TLR4could physically interact with CTSV mutant with impaired activity (C138S, N292Q). Besides, given at reperfusion time and without pretreatment, SQV could still reduce the liver ischemia/reperfusion injury.Conclusions:Thus, these studies confirm that CTSV is a target through which SQV blocks HMGB1-TLR4driven pathologic inflammation and demonstrate a potential role for cathepsin V in TLR4-MyD88recruitment. And the mechanism of action for SQV targeting TLR4receptor activation may involve a blockade of CTSV-TLR4interaction. The fact that CTSV inhibitors could target both TLR4and NF-κB level and show protection at a clinical relevantly time manner would allow for a rapid transition of the discovery to the bedside, by re-purposing a drug already approved by the FDA with a well-known safety profile or taking SQV as a lead to develop more tolerant and potent CTSV inhibitors for tissue injury protection/anti-inflammation.