The HIV protease inhibitor saquinavir inhibits HMGB1 driven inflammation by targeting the interaction of cathepsin V with TLR4/MyD88

Extracellular HMGB1 (disulfide form), via activation of Toll-Like-Receptor (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report, that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1 induced TLR4 activation, as assayed by the production of TNF-alpha by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334, or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver I/R, models both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4 driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside

Identification of Pharmacological Modulators of HMGB1-Induced Inflammatory Response by Cell-Based Screening

High mobility group box 1 (HMGB1), a highly conserved, ubiquitous protein, is released into the circulation during sterile inflammation (e.g. arthritis, trauma) and circulatory shock. It participates in the pathogenesis of delayed inflammatory responses and organ dysfunction. While several molecules have been identified that modulate the release of HMGB1, less attention has been paid to identify pharmacological inhibitors of the downstream inflammatory processes elicited by HMGB1 (C23-C45 disulfide C106 thiol form). In the current study, a cell-based medium-throughput screening of a 5000+ compound focused library of clinical drugs and drug-like compounds was performed in murine RAW264.7 macrophages, in order to identify modulators of HMGB1-induced tumor-necrosis factor alpha (TNFα) production. Clinically used drugs that suppressed HMGB1-induced TNFα production included glucocorticoids, beta agonists, and the anti-HIV compound indinavir. A re-screen of the NIH clinical compound library identified beta-agonists and various intracellular cAMP enhancers as compounds that potentiate the inhibitory effect of glucocorticoids on HMGB1-induced TNFα production. The molecular pathways involved in this synergistic anti-inflammatory effect are related, at least in part, to inhibition of TNFα mRNA synthesis via a synergistic suppression of ERK/IκB activation. Inhibition of TNFα production by prednisolone+salbutamol pretreatment was also confirmed in vivo in mice subjected to HMGB1 injection; this effect was more pronounced than the effect of either of the agents administered separately. The current study unveils several drug-like modulators of HMGB1-mediated inflammatory responses and offers pharmacological directions for the therapeutic suppression of inflammatory responses in HMGB1-dependent diseases. © 2013 Gerö et al

MD-2 is required for disulfide HMGB1-dependent TLR4 signaling

Innate immune receptors for pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) orchestrate inflammatory responses to infection and injury. Secreted by activated immune cells or passively released by damaged cells, HMGB1 is subjected to redox modification that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguished between HMGB1 isoforms. Here we demonstrate that the extracellular TLR4 adaptor, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide isoform of HMGB1, to the exclusion of other isoforms. Using MD-2–deficient mice, as well as MD-2 silencing in macrophages, we show a requirement for HMGB1-dependent TLR4 signaling. By screening HMGB1 peptide libraries, we identified a tetramer (FSSE, designated P5779) as a specific MD-2 antagonist preventing MD-2–HMGB1 interaction and TLR4 signaling. P5779 does not interfere with lipopolysaccharide-induced cytokine/chemokine production, thus preserving PAMP-mediated TLR4–MD-2 responses. Furthermore, P5779 can protect mice against hepatic ischemia/reperfusion injury, chemical toxicity, and sepsis. These findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms. The results may direct toward strategies aimed at attenuating DAMP-mediated inflammation while preserving antimicrobial immune responsiveness

Concentration- and time-dependence of the HMGB1-induced inflammatory response and reduction in cell viability in RAW 264.7 macrophages.

<p>RAW 264.7 cells were treated with the indicated amount of HMGB1 for 24, 48 or 72 hours. <b>A:</b> Cell viability was measured with the MTT assay and <b>B:</b> TNFα secretion was measured in the supernatant.</p

Prednisolone and salbutamol inhibit the HMGB-induced TNFα production.

<p>RAW 264.7 cells were pretreated with prednisolone (1 µM) and salbutamol (1 µM) and then exposed to HMGB1 (5 µg/ml) for various time up to 18 hours. <b>A</b>: TNFα secretion measured in the supernatant is plotted versus exposure length. (MEAN±SD values are shown) <b>B</b>: TNFα mRNA expression, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), is shown as fold expression values of vehicle treated cells. (CTL: vehicle treated control, HMGB: cells exposed to HMGB1, Pred: cells pretreated with prednisolone and exposed to HMGB1, Salb: cells pretreated with salbutamol and exposed to HMGB1, Pred+Salb: cells pretreated with both prednisolone and salbutamol and exposed to HMGB1. ^§p<0.05 HMGB1-treated group compared to vehicle treated control, *p<0.05 compared to HMGB1 group, ^#p<0.05 compared to single compound treatment).</p

Combined screening to identify pharmacological potentiators of dexamethasone-mediated inhibition of the HMGB1-induced pro-inflammatory response.

<p>RAW 264.7 cells were pre-treated with dexamethasone (3 µM) in combination with test compounds and exposed to HMGB1 for 18 hours. TNFα production was measured from the supernatant and the viability of the cells was measured by the MTT assay. <b>A:</b> TNFα responses measured in the combination screen are plotted versus the TNFα production values measured in the single compound screen. TNFα production values higher than MEAN+2SD are shown in red (“steroid inhibitors”) and values lower than MEAN+2SD in green boxes (“potentiators of steroids) for the combination screen. Red dots denote the toxic compounds, green the steroid potentiators and purple those that increase the TNFα production. Compounds that inhibited the HMGB-induced TNFα production in the single compound screen, but failed to potentiate the action of steroids are shown in yellow. <b>B:</b> TNFα responses relative to the activity of dexamethasone are plotted versus the viability values. Red and green boxes indicate the upper and lower 2 SD limits.</p

Screening for compounds that reduce the HMGB1-induced pro-inflammatory response.

<p><b>A:</b> Timeline of the cell-based screening: RAW 264.7 cells were pre-treated with test compounds and exposed to HMGB1 for 18 hours. TNFα production was measured from the supernatant and the viability of the cells was measured by the MTT assay. <b>B:</b> Dot graph showing the individual TNFα/viability results of the tested 5,646 compounds. TNFα responses are shown as % values of the HMGB1-induced TNFα production. Values lower than MEAN-2SD are shown in red (viability) and green (TNFα response) boxes to denote “toxic” and “Hit” compounds. <b>C–D:</b> Distribution of viability (C) and TNFα response (D) data with superimposed Gaussian distribution curves fitted to the data points.</p

HMGB1 induces time-dependent caspase activation in RAW 264.7 macrophages.

<p>RAW 264.7 cells were exposed to HMGB1 (5 µg/ml) for 24, 48 or 72 hours. Activated Caspase-3 was detected in cell extracts by Western blotting. Tubulin was used for loading control. The graph shows relative Caspase-3 activation values, normalized to tubulin. (**p<0.01 shows significant caspase activation compared to vehicle-treated cells).</p