Disclosed are compounds having the formula (I) wherein X, Y, Z1, Z2, Z3, Z4, R5, RA, m, A. L, and B are as defined herein, and methods of making and using the same.
Ramanjulu, Joshi M.
Pesiridis, G. Scott
Yang, Jingsong
Concha, Nestor
Singhaus, Robert
Zhang, Shu-Yun
Tran, Jean-Luc
Moore, Patrick
Lehmann, Stephanie
Eberl, H. Christian
Muelbaier, Marcel
Schneck, Jessica L.
Clemens, Jim
Adam, Michael
Mehlmann, John
Romano, Joseph
Morales, Angel
Kang, James
Leister, Lara
Graybill, Todd L.
Charnley, Adam K.
Ye, Guosen
Nevins, Neysa
Behnia, Kamelia
Wolf, Amaya I.
Kasparcova, Viera
Nurse, Kelvin
Wang, Liping
Li, Yue
Klein, Michael
Hopson, Christopher B.
Guss, Jeffrey
Bantscheff, Marcus
Bergamini, Giovanna
Reilly, Michael A.
Lian, Yiqian
Duffy, Kevin J.
Adams, Jerry
Foley, Kevin P.
Gough, Peter J.
Marquis, Robert W.
Smothers, James
Hoos, Axel
Bertin, John
Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA(1). The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants(2). To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery(3). Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immunemodifying cancer therapies.
Luz, Nivea Farias
Balaji, Sakthi
Okuda, Kendi
Barreto, Aline Silva
Bertin, John
Gough, Peter J.
Gazzinelli, Ricardo
Almeida, Roque P.
Bozza, Marcelo T.
Borges, Valeria M.
Chan, Francis Ka-Ming
Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1 beta expression in response to Leishmania. However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1b secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host.
Smethurst, Chris A.
Bevan, Nicola
Brooks, Carl
Emmons, Amanda
Gough, Peter J.
Mookherjee, Claudette
Moores, Kitty
Peace, Simon
Philp, Joanne
Piercy, Val
Watson, Steve P.
Zippoli, Mara
Optimisation of a series of biaryl sulphonamides resulted in the identification of compound 7 which demonstrated dose-dependent and strain-specific inhibition of monocyte recruitment in a thioglycollate-induced peritonitis model of inflammation. (C) 2012 Elsevier Ltd. All rights reserved.
Smethurst, Chris A.
Bevan, Nicola
Brooks, Carl
Emmons, Amanda
Gough, Peter J.
Mookherjee, Claudette
Moores, Kitty
Peace, Simon
Philp, Joanne
Piercy, Val
Watson, Steve P.
Zippoli, Mara
Optimisation of a series of biaryl sulphonamides resulted in the identification of compound 14 which demonstrated dose-dependent and strain-specific inhibition of monocyte recruitment in a thioglycollate-induced peritonitis model of inflammation.
Peterson, Lance W.
Philip, Naomi H.
Dillon, Christopher P.
Bertin, John
Gough, Peter J.
Green, Douglas R.
Brodsky, Igor E.
Innate immune responses that are crucial for control of infection are often targeted by microbial pathogens. Blockade of NF-kappa B and MAPK signaling by the Yersinia virulence factor YopJ inhibits cytokine production by innate immune cells but also triggers cell death. This cell death requires RIPK1 kinase activity and caspase-8, which are engaged by TLR4 and the adaptor protein TRIF. Nevertheless, TLR4-and TRIF-deficient cells undergo significant apoptosis, implicating TLR4/TRIF-independent pathways in the death of Yersinia-infected cells. In this article, we report a key role for TNF/TNFR1 in Yersinia-induced cell death of murine macrophages, which occurs despite the blockade of NF-kappa B and MAPK signaling imposed by Yersinia on infected cells. Intriguingly, direct analysis of YopJ injection revealed a heterogeneous population of injection-high and injection-low cells, and demonstrated that TNF expression came from the injection-low population. Moreover, TNF production by this subpopulation was necessary for maximal apoptosis in the population of highly injected cells, and TNFR-deficient mice displayed enhanced susceptibility to Yersinia infection. These data demonstrate an important role for collaboration between TNF and pattern recognition receptor signals in promoting maximal apoptosis during bacterial infection, and demonstrate that heterogeneity in virulence factor injection and cellular responses play an important role in promoting anti-Yersinia immune defense.
Najjar, Malek
Suebsuwong, Chalada
Ray, Soumya S.
Thapa, Roshan J.
Maki, Jenny L.
Nogusa, Shoko
Shah, Saumil
Saleh, Danish
Gough, Peter J.
Bertin, John
Yuan, Junying
Balachandran, Siddharth
Cuny, Gregory D.
Degterev, Alexei
RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-alpha) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective "hybrid'' RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.
Manda, Pratyusha
Feng, Yanjun
Lyons, John D.
Berger, Scott B.
Otani, Shunsuke
DeLaney, Alexandra
Tharp, Gregory K.
Maner-Smith, Kristal
Burd, Eileen M.
Schaeffer, Michelle
Hoffman, Sandra
Capriotti, Carol
Roback, Linda
Young, Cedrick B.
Liang, Zhe
Ortlund, Eric A.
DiPaolo, Nelson C.
Bosinger, Steven
Bertin, John
Gough, Peter J.
Brodsky, Igor E.
Coopersmith, Craig M.
Shayakhmetov, Dmitry M.
Mocarski, Edward S.
The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from cas pase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-beta-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.
Turner, Gregory H.
Olzinski, Alan R.
Bernard, Roberta E.
Aravindhan, Karpagam
Boyle, Ryan J.
Newman, Matt J.
Gardner, Scott D.
Willette, Robert N.
Gough, Peter J.
Jucker, Beat M.
Purpose: To evaluate the use of an ultrasmall superparamagnetic iron oxide (USPIO) contrast agent as a marker for the detection of macrophage in a preclinical abdominal aortic aneurysm animal (AAA) model. Materials and Methods: Osmotic pumps were implanted subcutaneously in apoE(-/-) mice for continuous infusion of Angiotensin II (Ang-II). Weekly bright-blood gradient echo scans were performed on the suprarenal abdominal aorta to evaluate aneurysm development. Once an AAA was detected, animals were administered 1000 mu mol/kg of the USPIO contrast agent ferumoxtran-10 (Combidex (R)) followed by in vivo scanning 24 h post-USPIO administration. After in vivo imaging, aortas were harvested for ex vivo imaging, histology, iron quantification, and gene expression analysis. Results: Reduced signal intensity was evident in the post-USPIO transverse images of the abdominal aorta. The areas of reduced signal were primarily along the aneurysm shoulder and outer perianeurysm areas and corresponded to regions of macrophage infiltration and colocalized USPIO determination by mew-is of histological staining. The absolute iron content measured significantly correlated to the area of signal reduction in the ex vivo images (r = 0.9; P < 0.01). In the AAA tissue, the macrophage-driven cytokine gene expression was up-regulated along with a matrix metalloproteinase known to mediate extracellular matrix breakdown in this disease model. Conclusion: These results demonstrate the feasibility of using an USPIO contrast agent as a surrogate for detecting the acute inflammatory process involved in the development of abdominal aneurysms.
Peterson, Lance W.
Philip, Naomi H.
DeLaney, Alexandra
Wynosky-Dolfi, Meghan A.
Asklof, Kendra
Gray, Falon
Choa, Ruth
Bjanes, Elisabet
Buza, Elisabeth L.
Hu, Baofeng
Dillon, Christopher P.
Green, Douglas R.
Berger, Scott B.
Gough, Peter J.
Bertin, John
Brodsky, Igor E.
Many pathogens deliver virulence factors or effectors into host cells in order to evade host defenses and establish infection. Although such effector proteins disrupt critical cellular signaling pathways, they also trigger specific antipathogen responses, a process termed "effector-triggered immunity." The Gram-negative bacterial pathogen Yersinia inactivates critical proteins of the NF-kappa B and MAPK signaling cascade, thereby blocking inflammatory cytokine production but also inducing apoptosis. Yersinia-induced apoptosis requires the kinase activity of receptor-interacting protein kinase 1 (RIPK1), a key regulator of cell death, NF-kappa B, and MAPK signaling. Through the targeted disruption of RIPK1 kinase activity, which selectively disrupts RIPK1-dependent cell death, we now reveal that Yersinia-induced apoptosis is critical for host survival, containment of bacteria in granulomas, and control of bacterial burdens in vivo. We demonstrate that this apoptotic response provides a cell-extrinsic signal that promotes optimal innate immune cytokine production and antibacterial defense, demonstrating a novel role for RIPK1 kinase-induced apoptosis in mediating effector-triggered immunity to circumvent pathogen inhibition of immune signaling.
Saleh, Danish
Najjar, Malek
Zelic, Matija
Shah, Saumil
Nogusa, Shoko
Polykratis, Apostolos
Paczosa, Michelle K.
Gough, Peter J.
Bertin, John
Whalen, Michael
Fitzgerald, Katherine A.
Slavov, Nikolai
Pasparakis, Manolis
Balachandran, Siddharth
Kelliher, Michelle
Mecsas, Joan
Degterev, Alexei
The innate immune response is a central element of the initial defense against bacterial and viral pathogens. Macrophages are key innate immune cells that upon encountering pathogen-associated molecular patterns respond by producing cytokines, including IFN-beta. In this study, we identify a novel role for RIPK1 and RIPK3, a pair of homologous serine/threonine kinases previously implicated in the regulation of necroptosis and pathologic tissue injury, in directing IFN-beta production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-beta synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase-dependent IFN-beta production may be elicited in an analogous fashion using LPS in bone marrow-derived macrophages upon inhibition of caspases. Notably, this regulation requires kinase activities of both RIPK1 and RIPK3, but not the necroptosis effector protein, MLKL. Mechanistically, we provide evidence that necrosome-like RIPK1 and RIPK3 aggregates facilitate canonical TRIF-dependent IFN-beta production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase-dependent synthesis of IFN-beta is markedly induced by avirulent strains of Gram-negative bacteria, Yersinia and Klebsiella, and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-beta during the host inflammatory responses to bacterial infection and suggest that the axis in which these kinases operate may represent a target for bacterial virulence factors.
Wang, Gren Z.
Haile, Pamela A.
Daniel, Tom
Belot, Benjamin
Viet, Andrew Q.
Goodman, Krista B.
Sha, Deyou
Dowdell, Sarah E.
Varga, Norbert
Hong, Xuan
Chakravorty, Subhas
Webb, Christine
Cornejo, Carla
Olzinski, Alan
Bernard, Roberta
Evans, Christopher
Emmons, Amanda
Briand, Jacques
Chung, Chun-Wa
Quek, Ruben
Lee, Dennis
Gough, Peter J.
Sehon, Clark A.
A series of biarylsulfonamides was identified as hCCR2 receptor antagonist but suffered from high plasma protein binding resulting in a >100 fold shift in activity in a functional GTP gamma S assay run in tandem in the presence and absence of human serum albumin. Introduction of an aryl amide with ethylenediamine linker led to compounds with reduced shifts and improved activity in whole blood. (C) 2011 Elsevier Ltd. All rights reserved.
Akey aspect of the innate immune system is the ability to discriminate between self and infectious nonself. This is achieved through pattern recognition receptors which directly recognise molecular epitopes expressed by microbes. Scavenger receptors (SRs) have been studied primarily due to their ability to bind and internalise modified lipoproteins, suggesting an important role in foam cell formation and the pathogenesis of atherosclerosis. However, the ability of some SRs to function as pattern recognition receptors through their binding of a wide variety of pathogens indicates a potential role in host defence. This review will detail our current understanding of the function of SRs in innate immunity, and in the initiation of aquired immune responses.
A key aspect of the innate immune system is the ability to discriminate between self and infectious nonself. This is achieved through pattern recognition receptors which directly recognise molecular epitopes expressed by microbes. Scavenger receptors (SRs) have been studied primarily due to their ability to bind and internalise modified lipoproteins, suggesting an important role in foam cell formation and the pathogenesis of atherosclerosis. However, the ability of some SRs to function as pattern recognition receptors through their binding of a wide variety of pathogens indicates a potential role in host defence. This review will detail our current understanding of the function of SRs in innate immunity, and in the initiation of aquired immune responses.
Liccardi, Gianmaria
Ramos Garcia, Laura
Tenev, Tencho
Annibaldi, Alessandro
Legrand, Arnaud J.
Robertson, David
Feltham, Rebecca
Anderton, Holly
Darding, Maurice
Peltzer, Nieves
Dannappel, Marius
Schünke, Hannah
Fava, Luca L.
Haschka, Manuel D.
Glatter, Timo
Nesvizhskii, Alexey
Schmidt, Alexander
Harris, Philip A.
Bertin, John
Gough, Peter J.
Villunger, Andreas
Silke, John
Pasparakis, Manolis
Bianchi, Katiuscia
Meier, Pascal
Zille, Marietta
Karuppagounder, Saravanan S.
Chen, Yingxin
Gough, Peter J.
Bertin, John
Finger, Joshua
Milner, Teresa A.
Jonas, Elizabeth A.
Ratan, Rajiv R.
Background and Purpose-Intracerebral hemorrhage leads to disability or death with few established treatments. Adverse outcomes after intracerebral hemorrhage result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options. Methods-We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were used. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy. Results-Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy, or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased after intracerebral hemorrhage in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by >80% and had similar therapeutic windows in vitro. Conclusions-Experimental intracerebral hemorrhage shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either of these pathways can bring cells below that threshold to survival.
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