Robert C. Reid

Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll‐like receptor 4 agonist LPS

Broad‐spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti‐ and proinflammatory effects on macrophages in vitro. We report here that several broad‐spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS‐induced mRNA expression of the proinflammatory mediators Edn‐1, Ccl‐7/MCP‐3, and Il‐12p40 but amplified the expression of the proatherogenic factors Cox‐2 and Pai‐1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS‐stimulated TEPM and HMDM. The pro‐ and anti‐inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1‐selective inhibitor, MS‐275, retained proinflammatory effects (amplification of LPS‐induced expression of Cox‐2 and Pai‐1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6‐selective inhibitor) retained anti‐inflammatory but not proinflammatory properties. Despite this, HDAC6−/− macrophages showed normal LPS‐induced expression of HDAC‐dependent inflammatory genes, arguing that the anti‐inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.

A Potent Human C5a Receptor Antagonist Protects against Disease Pathology in a Rat Model of Inflammatory Bowel Disease

The complement system is implicated in the pathogenesis of human inflammatory bowel disease, but the specific role of C5a has never been examined. We have compared the efficacy of an orally active human C5a receptor antagonist (AcPhe[Orn-Pro-d-cyclohexylalanine-Trp-Arg]), prednisolone, and infliximab against trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. The drugs were administered either 2 days before or 24 h after TNBS instillation, and rats were then examined after 8 days. Drug-free colitis control rats showed severe disease pathology with significant mortality (39%). Rats pre or posttreated with the C5a antagonist (10 mg/kg/day peroral, 0.3 mg/kg/day s.c.) had reduced mortality and significantly improved macroscopic scores, colon edema, colon myeloperoxidase levels, reduced concentrations of TNF-α levels in the colon and serum, and had greater food intake resulting in greater weight gains than colitis-only rats. Rats pretreated with prednisolone (1 mg/kg/day s.c.) displayed significant improvement in parameters measured, but posttreatment was ineffective. Single dose pretreatment with the TNF-α inhibitor infliximab (3 mg/kg i.v.) also had significant improvements in the parameters measured. Rats pretreated with a combination of the C5a antagonist and prednisolone showed no greater improvements than either drug alone. These findings suggest a central role for complement, particularly C5a, in the pathology of TNBS-induced colitis in rats, indicating a possible therapeutic role for C5a antagonists in inflammatory bowel disease.

Potencies of Human Immunodeficiency Virus Protease Inhibitors In Vitro against Plasmodium falciparum and In Vivo against Murine Malaria

ABSTRACT Parasite resistance to antimalarial drugs is a serious threat to human health, and novel agents that act on enzymes essential for parasite metabolism, such as proteases, are attractive targets for drug development. Recent studies have shown that clinically utilized human immunodeficiency virus (HIV) protease inhibitors can inhibit the in vitro growth of Plasmodium falciparum at or below concentrations found in human plasma after oral drug administration. The most potent in vitro antimalarial effects have been obtained for parasites treated with saquinavir, ritonavir, or lopinavir, findings confirmed in this study for a genetically distinct P. falciparum line (3D7). To investigate the potential in vivo activity of antiretroviral protease inhibitors (ARPIs) against malaria, we examined the effect of ARPI combinations in a murine model of malaria. In mice infected with Plasmodium chabaudi AS and treated orally with ritonavir-saquinavir or ritonavir-lopinavir, a delay in patency and a significant attenuation of parasitemia were observed. Using modeling and ligand docking studies we examined putative ligand binding sites of ARPIs in aspartyl proteases of P. falciparum (plasmepsins II and IV) and P. chabaudi (plasmepsin) and found that these in silico analyses support the antimalarial activity hypothesized to be mediated through inhibition of these enzymes. In addition, in vitro enzyme assays demonstrated that P. falciparum plasmepsins II and IV are both inhibited by the ARPIs saquinavir, ritonavir, and lopinavir. The combined results suggest that ARPIs have useful antimalarial activity that may be especially relevant in geographical regions where HIV and P. falciparum infections are both endemic.

Antifibrotic activity of an inhibitor of histone deacetylases in DOCA-salt hypertensive rats

Background and purpose:  Histone deacetylases (HDACs) silence genes by deacetylating lysine residues in histones and other proteins. HDAC inhibitors represent a new class of compounds with anti‐inflammatory activity. This study investigated whether treatment with a broad spectrum HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), would prevent cardiac fibrosis, part of the cardiovascular remodelling in deoxycorticosterone acetate (DOCA)‐salt rats.

Novel agonists and antagonists for human protease activated receptor 2.

Human protease activated receptor 2 (PAR2) is a G protein-coupled receptor that is associated with inflammatory diseases and cancers. PAR2 is activated by serine proteases that cleave its N-terminus and by synthetic peptides corresponding to the new N-terminus. Peptide agonists are widely used to characterize physiological roles for PAR2 but typically have low potency (e.g., SLIGKV-NH(2), SLIGRL-NH(2)), uncertain target selectivity, and poor bioavailability, limiting their usefulness for specifically interrogating PAR2 in vivo. Structure-activity relationships were used to derive new PAR2 agonists and antagonists containing nonpeptidic moieties. Agonist GB110 (19, EC(50) 0.28 μM) selectively induced PAR2-, but not PAR1-, mediated intracellular Ca(2+) release in HT29 human colorectal carcinoma cells. Antagonist GB83 (36, IC(50) 2 μM) is the first compound at micromolar concentrations to reversibly inhibit PAR2 activation by both proteases and other PAR2 agonists (e.g., trypsin, 2f-furoyl-LIGRLO-NH(2), 19). The new compounds are selective for PAR2 over PAR1, serum stable, and suitable for modulating PAR2 in disease models.

Comparative anti-inflammatory activities of antagonists to C3a and C5a receptors in a rat model of intestinal ischaemia/reperfusion injury

Complement activation is implicated in the pathogenesis of intestinal ischaemia–reperfusion injury (I/R), although the relative importance of individual complement components is unclear. A C3a receptor antagonist N(2)‐[(2,2‐diphenylethoxy)acetyl]‐L‐arginine (C3aRA) has been compared with a C5a receptor antagonist (C5aRA), AcF‐[OPdChaWR], in a rat model of intestinal I/R. C3aRA (IC50=0.15 μM) and C5aRA (IC50=0.32 μM) bound selectively to human polymorphonuclear leukocyte (PMN) C3a and C5a receptors, respectively. Effects on circulating neutrophils and blood pressure in the rat were also assessed. Anaesthetised rats, subjected to intestinal ischaemia (30 min) and reperfusion (120 min), were administered intravenously with either (A) the C3aRA (0.1–1.0 mg kg−1); the C5aRA (1.0 mg kg−1); the C3aRA+C5aRA (each 1.0 mg kg−1); or vehicle, 45 min prior, or (B) the C3aRA (1.0 mg kg−1) or vehicle, 120 min prior to reperfusion. The C3aRA and C5aRA, administered 45 min prior to reperfusion, displayed similar efficacies at ameliorating several disease markers (increased oedema, elevated ALT levels and mucosal damage) of rat intestinal I/R. The combination drug treatment did not result in greater injury reduction than either antagonist alone. However, doses of the C3aRA (0.01–10 mg kg−1) caused transient neutropaenia, and the highest dose (10 mg kg−1) also caused a rapid and transient hypertension. The C3aRA (1.0 mg kg−1), delivered 120 min prior to reperfusion to remove the global effect of C3aRA‐induced neutrophil sequestration, did not attenuate the markers of intestinal I/R, despite persistent C3aR antagonism at this time. C3aR antagonism does not appear to be responsible for the anti‐inflammatory actions of this C3aRA in intestinal I/R in the rat. Instead, C3aRA‐mediated global neutrophil tissue sequestration during ischaemia and early reperfusion may account for the protective effects observed.

C5aR and C3aR antagonists each inhibit diet-induced obesity, metabolic dysfunction, and adipocyte and macrophage signaling

Mammalian survival depends on metabolizing nutrients, storing energy, and combating infection. Complement activation in blood triggers energy‐depleting immune responses to fight infections. Here we identify surprising energy‐conserving roles for complement proteins C5a and C3a and their receptors, C5aR and C3aR, roles that are contraindicated in complement biology. Rats fed a high‐carbohydrate high‐fat diet developed obesity, visceral adiposity, adipose inflammation, glucose/insulin intolerance, and cardiovascular dysfunction that correlated with increased plasma C3a, adipose C5aR, and C3aR. These in vivo changes were dramatically attenuated by receptor‐selective antagonists of either C5aR (5 mg/kg/d p.o.) or C3aR (30 mg/kg/d p.o.), which both reduced proinflammatory adipokines and altered expression of inflammatory genes in adipose tissue. In vitro C5a and C3a (100 nM) exhibited novel insulin‐like effects on 3T3‐L1 adipocytes, promoting energy conservation by increasing glucose and fatty acid uptake while inhibiting cAMP signaling and lipolysis, and induced PGE2 release from macrophages, effects all blocked by each respective antagonist (10 μM). These studies reveal important new links between complement signaling and metabolism, highlight new complement functions on adipocytes and in adipose tissue, demonstrate how aberrant immune responses may exacerbate obesity and metabolic dysfunction, and show that targeting C3aR or C5aR with antagonists is a new strategy for treating metabolic dysfunction.—Lim, J., Iyer, A., Suen, J. Y., Seow, V., Reid, R. C., Brown, L., Fairlie, D. P. C5aR and C3aR antagonists each inhibit diet‐induced obesity, metabolic dysfunction, and adipocyte and macrophage signaling. FASEB J. 27, 822–831 (2013). www.fasebj.org

D-Tyrosine as a chiral precusor to potent inhibitors of human nonpancreatic secretory phospholipase A2 (IIa) with antiinflammatory activity.

Few reported inhibitors of secretory phospholipase A2 enzymes truly inhibit the IIa human isoform (hnpsPLA2‐IIa) noncovalently at submicromolar concentrations. Herein, the simple chiral precursor D‐tyrosine was derivatised to give a series of potent new inhibitors of hnpsPLA2‐IIa. A 2.2‐Å crystal structure shows an inhibitor bound in the active site of the enzyme, chelated to a Ca2+ ion through carboxylate and amide oxygen atoms, H‐bonded through an amide NH group to His48, with multiple hydrophobic contacts and a T‐shaped aromatic‐group–His6 interaction. Antiinflammatory activity is also demonstrated for two compounds administered orally to rats.

Inhibition of immune‐complex mediated dermal inflammation in rats following either oral or topical administration of a small molecule C5a receptor antagonist

Initiation of a peritoneal Arthus reaction by deposition of immune‐complexes results in vascular leakage, polymorphonuclear leukocyte (PMN) infiltration, and tumour necrosis factor α (TNFα) and interleukin‐6 (IL‐6) production. We now demonstrate in rats that oral administration of the C5a receptor antagonist AcPhe[Orn‐Pro‐D‐Cyclohexylalanine‐Trp‐Arg] (AcF‐[OPdChaWR]; 1 – 10 mg kg−1 30 min prior to immune‐complex deposition) inhibits these inflammatory markers in the peritoneal Arthus reaction. Initiation of a dermal Arthus reaction resulted in a significant increase in vascular leakage, PMN infiltration, systemic production of TNFα and pathological changes in the dermis. Pretreatment of rats with AcF‐[OPdChaWR] either intravenously (1 mg kg−1 10 min prior to immune‐complex deposition) or orally (1 – 10 mg kg−1 30 min prior to immune‐complex deposition) significantly inhibited immune‐complex mediated dermal vascular leakage and systemic cytokine production. Topical pretreatment with AcF‐[OPdChaWR] (400 μg site−1 in 10% dimethyl sulphoxide 10 min prior to immune‐complex deposition) also inhibited vascular leakage, as well as histopathological changes associated with a dermal Arthus reaction. Oral administration of 3 mg kg−1 AcF‐[OPdChaWR] resulted in the appearance of the drug in plasma within 5 min, with peak blood levels ∼0.3 μM reached within 20 min. The plasma elimination half‐life was ∼70 min. The oral activity and bioavailability of AcF‐[OPdChaWR], its activity when applied topically to the skin, suggest that small molecule C5a receptor antagonists may have therapeutic utility in dermal inflammatory disorders involving complement activation. This is the first demonstration for either an orally or topically active C5a receptor antagonist, and suggests that small molecule C5a antagonists may have therapeutic utility when given by multiple routes of application.

Comparative protection against rat intestinal reperfusion injury by a new inhibitor of sPLA2, COX‐1 and COX‐2 selective inhibitors, and an LTC4 receptor antagonist

A new group IIa sPLA2 inhibitor was compared with selective inhibitors of COX‐1, COX‐2 and an LTC4 antagonist for effects on local and remote tissue injuries following ischaemia and reperfusion (I/R) of the small intestine in rats. In an acute model of ischaemia (30 min) and reperfusion (150 min) injury in the absence of inhibitors, there was significant intestinal haemorrhage, oedema and mucosal damage, neutropenia, elevated serum levels of aspartate aminotransferase (AST) and hypotension. Preischaemic treatment with the inhibitor of sPLA2 (Group IIa), at 5 mg kg−1 i.v. or 10 mg kg−1 p.o. significantly inhibited I/R‐induced neutropenia, the elevation of serum levels of AST, intestinal oedema and hypotension. Pretreatment with the COX‐2 inhibitor celebrex (10 mg kg−1 i.v.) and the LTC4 antagonist zafirlukast (1 mg kg−1 i.v.) also showed marked improvement with I/R‐induced AST, oedema and neutropenia. Hypotension was only reduced by the LTC4 antagonist. The COX‐1 inhibitor flunixin (1 mg kg−1 i.v.) did not effect improvement in the markers of tissue injury. Histological examination of rat I/R injury showed that all of the drugs offered some protection to the mucosal layer damage compared to no drug treatment. Given i.v., the sPLA2 inhibitor was more effective than either the COX‐1 or COX‐2 inhibitors in preventing rat I/R injury. These results indicate that a potent new inhibitor of sPLA2 (group IIa) protects the rat small intestine from I/R injury after oral or intravenous administration. COX‐2 and LTC4 inhibitors also showed some beneficial effects against intestinal I/R injury. Our study suggests that sPLA2 (Group IIa) may have a pathogenic role in intestinal I/R in rats.