Azapeptides -A History of Synthetic Milestones and Key Examples.

For over 50 years of azapeptide synthetic techniques, developments have renewed the field of peptidomimetic therapeutics. Azapeptides are close surrogates of natural peptides: they contain a substitution of the amino acid α-carbon by a nitrogen atom. Goserelin (1989) and Atazanavir (2003) are two well-known, FDA-approved azapeptide-based drugs for the treatment of cancers and HIV infection, providing evidence for the successful clinical implementation of this class of therapeutic. This review highlights the azapeptides in recent medicinal chemistry applications and synthetic milestones. We describe the current techniques for azapeptide bond formation by introducing azapeptide coupling reagents and chain elongation methods both in solution and solid-phase strategies.

ß2-spectrin (SPTBN1) as a therapeutic target for diet-induced liver disease and preventing cancer development.

The prevalence of nonalcoholic steatohepatitis (NASH) and liver cancer is increasing. De novo lipogenesis and fibrosis contribute to disease progression and cancerous transformation. Here, we found that ß2-spectrin (SPTBN1) promotes sterol regulatory element (SRE)­binding protein (SREBP)­stimulated lipogenesis and development of liver cancer in mice fed a high-fat diet (HFD) or a western diet (WD). Either hepatocyte-specific knockout of SPTBN1 or siRNA-mediated therapy protected mice from HFD/WD-induced obesity and fibrosis, lipid accumulation, and tissue damage in the liver. Biochemical analysis suggested that HFD/WD induces SPTBN1 and SREBP1 cleavage by CASPASE-3 and that the cleaved products interact to promote expression of genes with sterol response elements. Analysis of human NASH tissue revealed increased SPTBN1 and CASPASE-3 expression. Thus, our data indicate that SPTBN1 represents a potential target for therapeutic intervention in NASH and liver cancer.

A Nonlethal Murine Flame Burn Model Leads to a Transient Reduction in Host Defenses and Enhanced Susceptibility to Lethal Pseudomonas aeruginosa Infection.

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host's susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1ß, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h (P < 0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in proinflammatory cytokines, is associated with postburn mortality.

Redox modifications of cysteine residues regulate the cytokine activity of HMGB1.

BACKGROUND: High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is passively released during cell death and secreted by activated cells of many lineages. HMGB1 contains three conserved redox-sensitive cysteine residues: cysteines in position 23 and 45 (C23 and C45) can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. METHODS: Primary human macrophages or murine macrophage-like RAW 264.7 cells were activated in cell cultures by redox-modified or point-mutated (C45A) recombinant HMGB1 preparations or by lipopolysaccharide (E. coli.0111: B4). Cellular phosphorylated NF-κB p65 subunit and subsequent TNF-α release were quantified by commercial enzyme-linked immunosorbent assays. RESULTS: Cell cultures with primary human macrophages and RAW 264.7 cells demonstrated that fully reduced HMGB1 with all three cysteines expressing thiol side chains failed to generate phosphorylated NF-КB p65 subunit or TNF-α. Mild oxidation forming a C23-C45 disulfide bond, while leaving C106 with a thiol group, was required for HMGB1 to induce phosphorylated NF-КB p65 subunit and TNF-α production. The importance of a C23-C45 disulfide bond was confirmed by mutation of C45 to C45A HMGB1, which abolished the ability for cytokine induction. Further oxidation of the disulfide isoform also inactivated HMGB1. CONCLUSIONS: These results reveal critical post-translational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during inflammation.

Human Dermcidin Protects Mice Against Hepatic Ischemia-Reperfusion-Induced Local and Remote Inflammatory Injury.

Background: Hepatic ischemia and reperfusion (I/R) injury is commonly associated with surgical liver resection or transplantation, and represents a major cause of liver damage and graft failure. Currently, there are no effective therapies to prevent hepatic I/R injury other than ischemic preconditioning and some preventative strategies. Previously, we have revealed the anti-inflammatory activity of a sweat gland-derived peptide, dermcidin (DCD), in macrophage/monocyte cultures. Here, we sought to explore its therapeutic potential and protective mechanisms in a murine model of hepatic I/R. Methods: Male C57BL/6 mice were subjected to hepatic ischemia by clamping the hepatic artery and portal vein for 60 min, which was then removed to initiate reperfusion. At the beginning of reperfusion, 0.2 ml saline control or solution of DCD (0.5 mg/kg BW) or DCD-C34S analog (0.25 or 0.5 mg/kg BW) containing a Cys (C)→Ser (S) substitution at residue 34 was injected via the internal jugular vein. For survival experiments, mice were subjected to additional resection to remove non-ischemic liver lobes, and animal survival was monitored for 10 days. For mechanistic studies, blood and tissue samples were collected at 24 h after the onset of reperfusion, and subjected to measurements of various markers of inflammation and tissue injury by real-time RT-PCR, immunoassays, and histological analysis. Results: Recombinant DCD or DCD-C34S analog conferred a significant protection against lethal hepatic I/R when given intravenously at the beginning of reperfusion. This protection was associated with a significant reduction in hepatic injury, neutrophilic CXC chemokine (Mip-2) expression, neutrophil infiltration, and associated inflammation. Furthermore, the administration of DCD also resulted in a significant attenuation of remote lung inflammatory injury. Mechanistically, DCD interacted with epidermal growth factor receptor (EGFR), a key regulator of liver inflammation, and significantly inhibited hepatic I/R-induced phosphorylation of EGFR as well as a downstream signaling molecule, protein kinase B (AKT). The suppression of EGFR expression by transducing Egfr-specific shRNA plasmid into macrophages abrogated the DCD-mediated inhibition of nitric oxide (NO) production induced by a damage-associated molecular pattern (DAMP), cold-inducible RNA-binding protein, CIRP. Conclusions: The present study suggests that human DCD and its analog may be developed as novel therapeutics to attenuate hepatic I/R-induced inflammatory injury possibly by impairing EGFR signaling.

Evidence supporting the use of peptides and peptidomimetics as potential SARS-CoV-2 (COVID-19) therapeutics.

During a disease outbreak/pandemic situation such as COVID-19, researchers are in a prime position to identify and develop peptide-based therapies, which could be more rapidly and cost-effectively advanced into a clinical setting. One drawback of natural peptide drugs, however, is their proteolytic instability; peptidomimetics can help to overcome this caveat. In this review, we summarize peptide and peptide-based therapeutics that target one main entry pathway of SARS-CoV-2, which involves the host ACE2 receptor and viral spike (S) protein interaction. Furthermore, we discuss the advantages of peptidomimetics and other potential targets that have been studied using peptide-based therapeutics for COVID-19.

Extracellular microRNA 130b-3p inhibits eCIRP-induced inflammation.

Although microRNAs regulate mRNA expression intracellularly, they are often released into the circulation in inflammatory diseases. During sepsis, secreted extracellular cold-inducible RNA-binding protein (eCIRP) acts as a damage-associated molecular pattern (DAMP), inducing tissue damage by elevating inflammatory cytokines and chemokines. Here, we report that the circulating microRNA 130b-3p inhibits eCIRP-mediated sterile and cecal ligation and puncture (CLP)-induced non-sterile inflammation. We find that levels of miR-130b-3p are increased in the serum of septic mice and patients and that it strongly interacts with recombinant murine (rm) CIRP in vitro and with eCIRP in the serum of septic mice in vivo. Combining a miR-130b-3p mimic with rmCIRP significantly decreases TNF-α release by macrophages compared to only rmCIRP-treated cells. This combined treatment also dose-dependently decreases the affinity of rmCIRP with its receptor TLR4/MD2. Finally, injection of a miR-130b-3p mimic significantly reduces rmCIRP- or CLP-induced systemic inflammation and acute lung injury in mice. These data show that extracellular miR-130b-3p functions as a novel endogenous inhibitor of eCIRP and point to an innovative therapeutic approach to treat inflammatory diseases.

Endogenous retroviruses are associated with hippocampus-based memory impairment.

Retrotransposons compose a staggering 40% of the mammalian genome. Among them, endogenous retroviruses (ERV) represent sequences that closely resemble the proviruses created from exogenous retroviral infection. ERVs make up 8 to 10% of human and mouse genomes and range from evolutionarily ancient sequences to recent acquisitions. Studies in Drosophila have provided a causal link between genomic retroviral elements and cognitive decline; however, in mammals, the role of ERVs in learning and memory remains unclear. Here we studied 2 independent murine models for ERV activation: muMT strain (lacking B cells and antibody production) and intracerebroventricular injection of streptozotocin (ICVI-STZ). We conducted behavioral assessments (contextual fear memory and spatial learning), as well as gene and protein analysis (RNA sequencing, PCR, immunohistochemistry, and western blot assays). Mice lacking mitochondrial antiviral-signaling protein (MAVS) and mice lacking stimulator of IFN genes protein (STING), 2 downstream sensors of ERV activation, provided confirmation of ERV impact. We found that muMT mice and ICVI-STZ mice induced hippocampal ERV activation, as shown by increased gene and protein expression of the Gag sequence of the transposable element intracisternal A-particle. ERV activation was accompanied by significant hippocampus-related memory impairment in both models. Notably, the deficiency of the MAVS pathway was protective against ICVI-STZ-induced cognitive pathology. Overall, our results demonstrate that ERV activation is associated with cognitive impairment in mice. Moreover, they provide a molecular target for strategies aimed at attenuating retroviral element sensing, via MAVS, to treat dementia and neuropsychiatric disorders.

Inhibition of HMGB1/RAGE-mediated endocytosis by HMGB1 antagonist box A, anti-HMGB1 antibodies, and cholinergic agonists suppresses inflammation.

BACKGROUND: Extracellular high mobility group box 1 protein  (HMGB1) serves a central role in inflammation as a transporter protein, which binds other immune-activating molecules that are endocytosed via the receptor for advanced glycation end-products (RAGE). These pro-inflammatory complexes are targeted to the endolysosomal compartment, where HMGB1 permeabilizes the lysosomes. This enables HMGB1-partner molecules to avoid degradation, to leak into the cytosol, and to reach cognate immune-activating sensors. Lipopolysaccharide (LPS) requires this pathway to generate pyroptosis by accessing its key cytosolic receptors, murine caspase 11, or the human caspases 4 and 5. This lytic, pro-inflammatory cell death plays a fundamental pathogenic role in gram-negative sepsis. The aim of the study was to identify molecules inhibiting HMGB1 or HMGB1/LPS cellular internalization. METHODS: Endocytosis was studied in cultured macrophages using Alexa Fluor-labeled HMGB1 or complexes of HMGB1 and Alexa Fluor-labeled LPS in the presence of an anti-HMGB1 monoclonal antibody (mAb), recombinant HMGB1 box A protein, acetylcholine, the nicotinic acetylcholine receptor subtype alpha 7 (α7 nAChR) agonist GTS-21, or a dynamin-specific inhibitor of endocytosis. Images were obtained by fluorescence microscopy and quantified by the ImageJ processing program (NIH). Data were analyzed using student's t test or one-way ANOVA followed by the least significant difference or Tukey's tests. RESULTS: Anti-HMGB1 mAb, recombinant HMGB1 antagonist box A protein, acetylcholine, GTS-21, and the dynamin-specific inhibitor of endocytosis inhibited internalization of HMGB1 or HMGB1-LPS complexes in cultured macrophages. These agents prevented macrophage activation in response to HMGB1 and/or HMGB1-LPS complexes. CONCLUSION: These results demonstrate that therapies based on HMGB1 antagonists and the cholinergic anti-inflammatory pathway share a previously unrecognized molecular mechanism of substantial clinical relevance.

Senescence as a main mechanism of Ritonavir and Ritonavir-NO action against melanoma.

The main focus of this study is exploring the effect and mechanism of two HIV-protease inhibitors: Ritonavir and Ritonavir-nitric oxide (Ritonavir-NO) on in vitro growth of melanoma cell lines. NO modification significantly improved the antitumor potential of Ritonavir, as the IC50 values of Ritonavir-NO were approximately two times lower than IC50 values of the parental compound. Our results showed for the first time, that both compounds induced senescence in primary and metastatic melanoma cell lines. This transformation was manifested as a change in cell morphology, enlargement of nuclei, increased cellular granulation, upregulation of ß-galactosidase activity, lipofuscin granules appearance, higher production of reactive oxygen species and persistent inhibition of proliferation. The expression of p53, as one of the key regulators of senescence, was upregulated after 48 hours of Ritonavir-NO treatment only in metastatic B16F10 cells, ranking it as a late-response event. The development of senescent phenotype was consistent with the alteration of the cytoskeleton-as we observed diminished expression of vinculin, α-actin, and ß-tubulin. Permanent inhibition of S6 protein by Ritonavir-NO, but not Ritonavir, could be responsible for a stronger antiproliferative potential of the NO-modified compound. Taken together, induction of senescent phenotype may provide an excellent platform for developing therapeutic approaches based on selective killing of senescent cells.

Lopinavir-NO, a nitric oxide-releasing HIV protease inhibitor, suppresses the growth of melanoma cells in vitro and in vivo.

We generated a nitric oxide (NO)-releasing derivative of the anti-HIV protease inhibitor lopinavir by linking the NO moiety to the parental drug. We investigated the effects of lopinavir and its derivative lopinavir-NO on melanoma cell lines in vitro and in vivo. Lopinavir-NO exhibited a twofold stronger anticancer action than lopinavir in vitro. These results were successfully translated into syngeneic models of melanoma in vivo, where a significant reduction in tumour volume was observed only in animals treated with lopinavir-NO. Both lopinavir and lopinavir-NO inhibited cell proliferation and induced the trans-differentiation of melanoma cells to Schwann-like cells. In melanoma cancer cell lines, both lopinavir and lopinavir-NO induced morphological changes, minor apoptosis and reactive oxygen species (ROS) production. However, caspase activation and autophagy were detected only in B16 cells, indicating a cell line-specific treatment response. Lopinavir-NO released NO intracellularly, and NO neutralization restored cell viability. Treatment with lopinavir-NO induced only a transient activation of Akt and inhibition of P70S6 kinase. The results of this study identify lopinavir-NO as a promising candidate for further clinical trials in melanoma and possibly other solid tumours.

Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex.

Macrophage cytokine production is regulated by neural signals, for example in the inflammatory reflex. Signals in the vagus and splenic nerves are relayed by choline acetyltransferase+ T cells that release acetylcholine, the cognate ligand for alpha7 nicotinic acetylcholine subunit-containing receptors (α7nAChR), and suppress TNF release in macrophages. Here, we observed that electrical vagus nerve stimulation with a duration of 0.1-60 s significantly reduced systemic TNF release in experimental endotoxemia. This suppression of TNF was sustained for more than 24 h, but abolished in mice deficient in the α7nAChR subunit. Exposure of primary human macrophages and murine RAW 264.7 macrophage-like cells to selective ligands for α7nAChR for 1 h in vitro attenuated TNF production for up to 24 h in response to endotoxin. Pharmacological inhibition of adenylyl cyclase (AC) and knockdown of adenylyl cyclase 6 (AC6) or c-FOS abolished cholinergic suppression of endotoxin-induced TNF release. These findings indicate that action potentials in the inflammatory reflex trigger a change in macrophage behavior that requires AC and phosphorylation of the cAMP response element binding protein (CREB). These observations further our mechanistic understanding of neural regulation of inflammation and may have implications for development of bioelectronic medicine treatment of inflammatory diseases.

Anticancer and Differentiation Properties of the Nitric Oxide Derivative of Lopinavir in Human Glioblastoma Cells.

Glioblastoma (GBM) is the most frequent and deadly form of primary malignant brain tumor among adults. A promising emerging approach for GBM treatment may be offered from HIV protease inhibitors (HIV-PIs). In fact, in addition to their primary pharmacological activity in the treatment of HIV infection, they possess important anti-neoplastic effects. According to previous studies, the addition of a nitric oxide (NO) donating group to parental compounds can reduce their toxicity and enhance the anticancer action of various compounds, including HIV-PIs. In this study we compared the effects of the HIV-PI Lopinavir (Lopi) and of its NO-derivative Lopinavir-NO (Lopi-NO) on the in vitro growth of LN-229 and U-251 human GBM cell lines. Lopi-NO reduced the viability of LN-229 and U-251 cells at significantly lower concentrations than the parental drug. In particular, Lopi-NO inhibited tumor cell proliferation and induced the differentiation of U-251 cells toward an astrocyte-like phenotype without triggering significant cell death in both cell types. The anticancer effect of Lopi-NO was persistent even upon drug removal. Furthermore, Lopi-NO induced strong autophagy that did not appear to be related to its chemotherapeutic action. Overall, our results suggest that Lopi-NO could be a potential effective anticancer drug for GBM treatment.

Folic acid derived-P5779 mimetics regulate DAMP-mediated inflammation through disruption of HMGB1:TLR4:MD-2 axes.

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) protein that mediates inflammatory responses after infection or injury. Previously, we reported a peptide inhibitor of HMGB1 (P5779) that acts by directly interrupting HMGB1/MD-2 binding. Here, fingerprint similarity search and docking studies suggest folic acid derived-drugs function as P5779 mimetopes. Molecular dynamic (MD) simulation studies demonstrate that folic acid mimics the binding of P5779 at the TLR4 and MD-2 intersection. In surface plasmon resonance (SPR) studies, these drugs showed direct binding to TLR4/MD-2 but not HMGB1. Furthermore, these P5779 mimetopes inhibit HMGB1 and MD-2 binding and suppress HMGB1-induced TNF release in human macrophages in the nanomolar range. We assert from our findings that their demonstrated anti-inflammatory effects may be working through TLR4-dependent signaling.

Connexin 43 Hemichannel as a Novel Mediator of Sterile and Infectious Inflammatory Diseases.

Cytoplasmic membrane-bound connexin 43 (Cx43) proteins oligomerize into hexameric channels (hemichannels) that can sometimes dock with hemichannels on adjacent cells to form gap junctional (GJ) channels. However, the possible role of Cx43 hemichannels in sterile and infectious inflammatory diseases has not been adequately defined due to the lack of selective interventions. Here we report that a proinflammatory mediator, the serum amyloid A (SAA), resembled bacterial endotoxin by stimulating macrophages to up-regulate Cx43 expression and double-stranded RNA-activated protein kinase R (PKR) phosphorylation in a TLR4-dependent fashion. Two well-known Cx43 mimetic peptides, the GAP26 and TAT-GAP19, divergently affected macrophage hemichannel activities in vitro, and differentially altered the outcome of lethal sepsis in vivo. By screening a panel of Cx43 mimetic peptides, we discovered that one cysteine-containing peptide, P5 (ENVCYD), effectively attenuated hemichannel activities, and significantly suppressed endotoxin-induced release of ATP and HMGB1 in vitro. In vivo, the P5 peptide conferred a significant protection against hepatic ischemia/reperfusion injury and lethal microbial infection. Collectively, these findings have suggested a pathogenic role of Cx43 hemichannels in sterile injurious as well as infectious inflammatory diseases possibly through facilitating extracellular ATP efflux to trigger PKR phosphorylation/activation.

High mobility group box 1 orchestrates tissue regeneration via CXCR4.

Inflammation and tissue regeneration follow tissue damage, but little is known about how these processes are coordinated. High Mobility Group Box 1 (HMGB1) is a nuclear protein that, when released on injury, triggers inflammation. We previously showed that HMGB1 with reduced cysteines is a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine. Here we report that fully reduced HMGB1 orchestrates muscle and liver regeneration via CXCR4, whereas disulfide HMGB1 and its receptors TLR4/MD-2 and RAGE (receptor for advanced glycation end products) are not involved. Injection of HMGB1 accelerates tissue repair by acting on resident muscle stem cells, hepatocytes, and infiltrating cells. The nonoxidizable HMGB1 mutant 3S, in which serines replace cysteines, promotes muscle and liver regeneration more efficiently than the wild-type protein and without exacerbating inflammation by selectively interacting with CXCR4. Overall, our results show that the reduced form of HMGB1 coordinates tissue regeneration and suggest that 3S may be used to safely accelerate healing after injury in diverse clinical contexts.

Activation of the cholinergic anti-inflammatory pathway by GTS-21 attenuates cisplatin-induced acute kidney injury in mice.

Acute kidney injury (AKI) is the most common side effect of cisplatin, a widely used chemotherapy drug. Although AKI occurs in up to one third of cancer patients receiving cisplatin, effective renal protective strategies are lacking. Cisplatin targets renal proximal tubular epithelial cells leading to inflammation, reactive oxygen species, tubular cell injury, and eventually cell death. The cholinergic anti-inflammatory pathway is a vagus nerve-mediated reflex that suppresses inflammation via α7 nicotinic acetylcholine receptors (α7nAChRs). Our previous studies demonstrated the renoprotective and anti-inflammatory effects of cholinergic agonists, including GTS-21. Therefore, we examined the effect of GTS-21 on cisplatin-induced AKI. Male C57BL/6 mice received either saline or GTS-21 (4mg/kg, i.p.) twice daily for 4 days before cisplatin and treatment continued through euthanasia; 3 days post-cisplatin mice were euthanized and analyzed for markers of renal injury. GTS-21 significantly reduced cisplatin-induced renal dysfunction and injury (p<0.05). GTS-21 significantly attenuated renal Ptgs2/COX-2 mRNA and IL-6, IL-1ß, and CXCL1 protein expression, as well as neutrophil infiltration after cisplatin. GTS-21 blunted cisplatin-induced renal ERK1/2 activation, as well as renal ATP depletion and apoptosis (p<0.05). GTS-21 suppressed the expression of CTR1, a cisplatin influx transporter and enhanced the expression of cisplatin efflux transporters MRP2, MRP4, and MRP6 (p<0.05). Using breast, colon, and lung cancer cell lines we showed that GTS-21 did not inhibit cisplatin's tumor cell killing activity. GTS-21 protects against cisplatin-AKI by attenuating renal inflammation, ATP depletion and apoptosis, as well as by decreasing renal cisplatin influx and increasing efflux, without impairing cisplatin-mediated tumor cell killing. Our results support further exploring the cholinergic anti-inflammatory pathway for preventing cisplatin-induced AKI.

Cathepsin L promotes Vascular Intimal Hyperplasia after Arterial Injury.

The inflammatory pathways that drive the development of intimal hyperplasia (IH) following arterial injury are not fully understood. We hypothesized that the lysosomal cysteine protease cathepsin L activates processes leading to IH after arterial injury. Using a mouse model of wire-induced carotid artery injury we showed that cathepsin L activity peaks at day 7 and remains elevated to 28 days. The genetic deletion of cathepsin L prevented IH and monocyte recruitment in the carotid wall. The injury-induced increases in cathepsin L mRNA and activity were mitigated in mice with myeloid-specific deletion of toll like receptor 4 (TLR4) or myeloid differentiation primary response gene 88 (MyD88). We further discovered that a HIV-protease inhibitor saquinavir (SQV), which is known to block recombinant mouse cathepsin L activity in vitro, prevented IH after arterial injury. SQV also suppressed LPS (TLR4 agonist) induced monocyte adhesion to endothelial monolayers. These findings establish cathepsin L as a critical regulator of the inflammation that leads to IH and that the TLR4- MyD88 pathway in myeloid lineages regulates cathepsin L expression in the vessel wall following wire injury. The FDA approved drug, SQV blocks IH though mechanisms that may include the suppression of cathepsin L.