Inhibition of Interleukin-23-Mediated Inflammation with a Novel Small Molecule Inverse Agonist of RORγt.

Blockade of interleukin (IL)-23 or IL-17 with biologics is clinically validated as a treatment of psoriasis. However, the clinical impact of targeting other nodes within the IL-23/IL-17 pathway, especially with small molecules, is less defined. We report on a novel small molecule inverse agonist of retinoid acid-related orphan receptor (ROR) γt and its efficacy in preclinical models of psoriasis and arthritis. 1-(2,4-Dichloro-3-((1,4-dimethyl-6-(trifluoromethyl)-1H-indol-2-yl)methyl)benzoyl)piperidine-4-carboxylic acid (A-9758) was optimized from material identified from a high-throughput screening campaign. A-9758 is selective for RORγt and exhibits robust potency against IL-17A release both in vitro and in vivo. In vivo, we also show that IL-23 is sufficient to drive the accumulation of RORγt+ cells, and inhibition of RORγt significantly attenuates IL-23-driven psoriasiform dermatitis. Therapeutic treatment with A-9758 (i.e., delivered during active disease) was also effective in blocking skin and joint inflammation. Finally, A-9758 exhibited efficacy in an ex vivo human whole blood assay, suggesting small molecule inverse agonists of RORγt could be efficacious in human IL-17-related diseases. SIGNIFICANCE STATEMENT: Using a novel small molecule inverse agonist, and preclinical assays, we show that RORγt is a viable target for the inhibition of RORγt/Th17-driven diseases such as psoriasis. Preclinical models of psoriasis show that inhibition of RORγt blocks both the accumulation and effector function of IL-17-producing T cells.

Discovery of novel quinoline sulphonamide derivatives as potent, selective and orally active RORγ inverse agonists.

A high-throughput screen against Inventiva's compound library using a Gal4/RORγ-LBD luciferase reporter gene assay led to the discovery of a new series of quinoline sulphonamides as RORγ inhibitors, eventually giving rise to a lead compound having an interesting in vivo profile after oral administration. This lead was evaluated in a target engagement model in mouse, where it reduced IL-17 cytokine production after immune challenge. It also proved to be active in a multiple sclerosis model (EAE) where it reduced the disease score. The synthesis, structure activity relationship (SAR) and biological activity of these derivatives is described herein.

The activation of p38 alpha, and not p38 beta, mitogen-activated protein kinase is required for ischemic preconditioning.

Numerous studies show that pharmacological inhibition of p38 mitogen-activated protein kinases (p38s) before lethal ischemia prevents conditioning. However, these inhibitors have off-target effects and do not discriminate between the alpha and beta isoforms; the activation of which is thought to have diverse and perhaps opposing actions with p38 alpha aggravating, and p38 beta reducing, myocardial injury. We adopted a chemical genetic approach using mice in which either the p38 alpha (DR alpha) or p38 beta (DR beta) alleles were targeted to substitute the "gatekeeper" threonine residue for methionine, thereby preventing the binding of a pharmacological inhibitor, SB203580. Isolated, perfused wild-type (WT), DR alpha and DR beta mouse hearts underwent ischemic preconditioning with 4 cycles of 4 min ischemia/6 min reperfusion, with or without SB203580 (10 microM), followed by 30 min of global ischemia and 120 min of reperfusion. In WT and DR beta hearts, SB203580 completely abolished the reduction in myocardial infarction seen with preconditioning and also the phosphorylation of downstream substrates of p38. These effects of SB203580 were not seen in DR alpha hearts. Furthermore ischemic preconditioning occurred unaltered in p38 beta null hearts. Contrary to expectation the activation of p38 alpha, and not p38 beta, is necessary for ischemic preconditioning. Since p38 alpha is also the isoform that leads to lethal myocardial injury, it is unlikely that targeted therapeutic strategies to achieve isoform-selective inhibition will only prevent the harmful consequences of activation.

A chemical genetic approach reveals that p38alpha MAPK activation by diphosphorylation aggravates myocardial infarction and is prevented by the direct binding of SB203580.

The use of nonselective pharmacological inhibitors has resulted in controversy regarding the mechanism and consequences of p38 activation during myocardial infarction. Classic p38 inhibitors such as SB203580 rely on a critical "gatekeeper" threonine residue for binding. We addressed these controversies by using mice in which the p38alpha alleles were targeted to cause substitution of the gatekeeper residue and resistance to inhibition. In homozygous drug-resistant compared with wild-type hearts, SB203580 failed to inhibit the activating phosphorylation of p38 or to reduce the infarction caused by myocardial ischemia. However, BIRB796, a p38 inhibitor not reliant on the gatekeeper for binding, similarly reduced p38-activating phosphorylation and infarction in both wild-type and knock-in mice, thereby excluding a nonspecific inhibitor-dependent phenotype resulting from the targeting strategy. Furthermore, the activation during myocardial ischemia involved phosphorylation of both the threonine and tyrosine residues in the activation loop of p38 despite the phosphorylation of the threonine alone being sufficient to create the epitope for dual phosphospecific antibody binding. Finally, SB203580 failed to reduce infarction in heterozygous drug-resistant hearts, suggesting that near complete inhibition of p38alpha kinase activity is necessary to elicit protection. These results indicate that, during myocardial ischemia, p38alpha (i) is the dominant-active p38 isoform, (ii) contributes to infarction, (iii) is responsible for the cardioprotective effect of SB203580, and (iv) is activated by a mechanism consistent with autodiphosphorylation despite this necessitating the phosphorylation of a tyrosine residue by an archetypal serine/threonine kinase.

Identification of cardiac myosin-binding protein C as a candidate biomarker of myocardial infarction by proteomics analysis.

Acute myocardial infarction (AMI) is a common cause of death for which effective treatments are available provided that diagnosis is rapid. The current diagnostic gold standards are circulating cardiac troponins I and T. However, their slow release delays diagnosis, and their persistence limits their utility in the identification of reinfarction. The aim was to identify candidate biomarkers of AMI. Isolated mouse hearts were perfused with oxygenated protein-free buffer, and coronary effluent was collected after ischemia or during matched normoxic perfusion. Effluents were analyzed using proteomics approaches based on one- or two-dimensional initial separation. Of the 459 proteins identified after ischemia with one-dimensional separation, 320 were not detected in the control coronary effluent. Among these were all classic existing biomarkers of AMI. We also identified the cardiac isoform of myosin-binding protein C in its full-length form and as a 40-kDa degradation product. This protein was not detected in the other murine organs examined, increased markedly with even trivial myocardial infarction, and could be detected in the plasma after myocardial infarction in vivo, a profile compatible with a biomarker of AMI. Two-dimensional fluorescence DIGE of ischemic and control coronary effluents identified more than 200 asymmetric spots verified by swapping dyes. Once again existing biomarkers of injury were confirmed as well as posttranslational modifications of antioxidant proteins such as peroxiredoxins. Perfusing hearts with protein-free buffers provides a platform of graded ischemic injury that allows detailed analysis of protein release and identification of candidate cardiac biomarkers like myosin-binding protein C.

Pharmacological postconditioning effect of muramyl dipeptide is mediated through RIP2 and TAK1.

AIMS: Despite their ability to cause septic shock and myocardial dysfunction, components of Gram-negative bacterial cell walls, like lipopolysaccharide, have been shown in numerous studies to induce myocardial protection during ischaemia-reperfusion injury. Muramyl dipeptide (MDP) is another such component recognized by an intracellular receptor, nucleotide-binding oligomerization domain 2. Receptor activation leads to intracellular signals through receptor interacting protein-2 (RIP2) and tumour growth factor-beta-activated kinase-1 (TAK1). However, little is known about the RIP2/TAK1 pathway in the heart. The aim of this study was to determine whether the RIP2/TAK1 pathway has a cardioprotective role in a mouse model of myocardial infarction. METHODS AND RESULTS: We isolated and subjected wild-type (WT) and RIP2(-/-) mouse hearts to 30 min of global ischaemia and 120 min of reperfusion with or without perfusion of MDP (10 microg/mL) before or after the ischaemic period and determined the infarct size. We examined activation of the TAK1/nuclear factor kappaB (NFkappaB) signalling pathway. The effect of TAK1 inhibition on MDP-induced cardioprotection was also evaluated. Exposure to MDP during reperfusion significantly reduced infarct size in WT hearts (from 51.7 +/- 5.6% in control to 38.1 +/- 6.7%, P < 0.05), but not in RIP2(-/-) hearts or in WT hearts with coincident pharmacological inhibition of TAK1. MDP treatment significantly increased the levels of p-TAK1 and p-JNK (Jun N-terminal kinase) and led to NFkappaB activation via phosphorylation and degradation of IkappaB in the WT, but not in the RIP2(-/-), myocardium. CONCLUSION: These results indicate that MDP at reperfusion induced cardioprotection through an RIP2/TAK1-dependent mechanism.

Glycogen synthase kinase-3 inactivation is not required for ischemic preconditioning or postconditioning in the mouse.

The inactivation of glycogen synthase kinase-3beta (GSK-3beta) is proposed as the event integrating protective pathways initiated by preconditioning and other interventions. The inactivation of GSK-3 is thought to decrease the probability of opening of the mitochondrial permeability transition pore. The aim of this study was to verify the role of GSK-3 using a targeted mouse line lacking the critical N-terminal serine within GSK-3beta (Ser9) and the highly homologous GSK-3alpha (Ser21), which when phosphorylated results in kinase inactivation. Postconditioning with 10 cycles of 5 seconds of reperfusion/5 seconds of ischemia and preconditioning with 6 cycles of 4 minutes of ischemia/6 minutes of reperfusion, similarly reduced infarction of the isolated perfused mouse heart in response to 30 minutes of global ischemia and 120 minutes of reperfusion. Preconditioning caused noticeable inactivating phosphorylation of GSK-3. However, both preconditioning and postconditioning still protected hearts of homozygous GSK-3 double knockin mice. Moreover, direct pharmacological inhibition of GSK-3 catalytic activity with structurally diverse inhibitors before or after ischemia failed to recapitulate conditioning protection. Nonetheless, cyclosporin A, a direct mitochondrial permeability transition pore inhibitor, reduced infarction in hearts from both wild-type and homozygous GSK-3 double knockin mice. Furthermore, in adult cardiac myocytes from GSK-3 double knockin mice, insulin exposure was still as effective as cyclosporin A in delaying mitochondrial permeability transition pore opening. Our results, which include a novel genetic approach, suggest that the inhibition of GSK-3 is unlikely to be the key determinant of cardioprotective signaling in either preconditioning or postconditioning in the mouse.

The role of RIP2 in p38 MAPK activation in the stressed heart.

The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinase-dead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.

The relationship between p38 mitogen-activated protein kinase and AMP-activated protein kinase during myocardial ischemia.

OBJECTIVE: p38 mitogen-activated protein kinase (p38 MAPK) and AMP-activated protein kinase (AMPK) are activated by, and influence sensitivity to, myocardial ischemia. Recently a number of studies have suggested that AMPK may participate in the activation of p38 MAPK. We therefore examined whether AMPK may be the principal "ischemia sensor" responsible for p38 MAPK activation during myocardial ischemia. METHODS: We used a variety of approaches to alter AMPK activity during ischemia and studied the repercussions on p38 MAPK activation. RESULTS: The activities of AMPK and p38 MAPK were temporally related in adult rat ventricular myocytes (ARVM) subjected to simulated ischemia and in isolated mouse hearts subjected to no-flow ischemia. However p38 MAPK activation was unaltered in mouse hearts lacking the predominant or minor myocardial isoforms, AMPKalpha2 or AMPKalpha1 respectively. Likewise, in ARVM, adenoviral-driven expression of the minor myocardial isoform AMPKalpha1, in a constitutively active or dominant negative form reducing AMPK activity, did not alter p38 MAPK activation under basal conditions or during simulated ischemia. Finally, pharmacological inhibition of AMPK during ischemia with compound C did not attenuate the coincident activation of p38 MAPK. CONCLUSIONS: Although AMPK and p38 MAPK are both activated during myocardial ischemia, the activation of p38 MAPK occurs independently of AMPK.

Activation of p38 mitogen-activated protein kinase contributes to the early cardiodepressant action of tumor necrosis factor.

OBJECTIVES: The purpose of this study was to determine whether p38 mitogen-activated protein kinase (p38-MAPK) contributes to tumor necrosis factor-alpha (TNFalpha)-induced contractile depression. BACKGROUND: Tumor necrosis factor has both beneficial and detrimental consequences that may result from the activation of different downstream pathways. Tumor necrosis factor activates p38-MAPK, a stress-responsive kinase implicated in contractile depression and cardiac injury. METHODS: In isolated hearts from mice lacking the p38-MAPK activator, MAPK kinase 3 (MKK3), perfused at constant coronary pressure or flow, we measured the left ventricular developed pressure (LVDP) and the relationship between end-diastolic volume and LVDP in the presence and absence of 10 ng/ml TNFalpha. RESULTS: Within 15 min at constant pressure, TNFalpha significantly reduced LVDP and coronary flow in outbred and mkk3(+/+) mice. This early negative inotropic effect was associated with a marked phosphorylation of both p38-MAPK and its indirect substrate, HSP27. In hearts lacking MKK3, TNFalpha failed to activate p38-MAPK or to cause significant contractile dysfunction. The actions of TNFalpha were similarly attenuated in MAPK-activated protein kinase 2 (MK2)-deficient hearts, which have a marked reduction in myocardial p38-MAPK protein content, and by the p38-MAPK catalytic site inhibitor SB203580 (1 micromol/l). Under conditions of constant coronary flow, the p38-MAPK activation and contractile depression induced by TNFalpha, though attenuated, remained sensitive to the absence of MKK3 or the presence of SB203580. The role of p38-MAPK in TNFalpha-induced contractile depression was confirmed in isolated murine cardiac myocytes exposed to SB203580 or lacking MKK3. CONCLUSIONS: Tumor necrosis factor activates p38-MAPK in the intact heart and in isolated cardiac myocytes through MKK3. This activation likely contributes to the early cardiodepressant action of TNFalpha.

Opioid receptor blockade reduces Fas-induced hepatitis in mice.

Fas (CD95)-induced hepatocyte apoptosis and cytotoxic activity of neutrophils infiltrating the injured liver are two major events leading to hepatitis. Because it has been reported that opioids, via a direct interaction, sensitize splenocytes to Fas-mediated apoptosis by upregulating Fas messenger RNA (mRNA) and modulated neutrophil activity, we assumed that opioids may participate in the pathophysiology of hepatitis. Using the hepatitis model induced by agonistic anti-Fas antibody in mice, we showed that opioid receptor blockade reduced liver damage and consequently increased the survival rate of animals when the antagonist naltrexone was injected simultaneously or prior to antibody administration. Treatment of mice with morphine enhanced mortality. Naloxone methiodide-a selective peripheral opioid antagonist-had a protective effect, but the absence of opioid receptors in the liver, together with lack of morphine effect in Fas-induced apoptosis of primary cultured hepatocytes, ruled out a direct effect of opioids on hepatocytes. In addition, the neutralization of opioid activity by naltrexone did not modify Fas mRNA expression in the liver as assessed with real-time quantitative polymerase chain reaction. Injured livers were infiltrated by neutrophils, but granulocyte-depleted mice were not protected against the enhancing apoptotic effect of morphine. In conclusion, opioid receptor blockade improves the resistance of mice to Fas-induced hepatitis via a peripheral mechanism that does not involve a down-modulation of Fas mRNA in hepatocytes nor a decrease in proinflammatory activity of neutrophils.

Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis.

The effect of high-density lipoprotein (HDL) in protecting against atherosclerosis is usually attributed to its role in 'reverse cholesterol transport'. In this process, HDL particles mediate the efflux and the transport of cholesterol from peripheral cells to the liver for further metabolism and bile excretion. Thus, cell-surface receptors for HDL on hepatocytes are chief partners in the regulation of cholesterol homeostasis. A high-affinity HDL receptor for apolipoprotein A-I (apoA-I) was previously identified on the surface of hepatocytes. Here we show that this receptor is identical to the beta-chain of ATP synthase, a principal protein complex of the mitochondrial inner membrane. Different experimental approaches confirm this ectopic localization of components of the ATP synthase complex and the presence of ATP hydrolase activity at the hepatocyte cell surface. Receptor stimulation by apoA-I triggers the endocytosis of holo-HDL particles (protein plus lipid) by a mechanism that depends strictly on the generation of ADP. We confirm this effect on endocytosis in perfused rat liver ex vivo by using a specific inhibitor of ATP synthase. Thus, membrane-bound ATP synthase has a previously unsuspected role in modulating the concentrations of extracellular ADP and is regulated by a principal plasma apolipoprotein.