Design and synthesis of new hybrid molecules that activate the transcription factor Nrf2 and simultaneously release carbon monoxide.

The transcription factor Nrf2 and its downstream target heme oxygenase-1 (HO-1) are essential protective systems against oxidative stress and inflammation. The products of HO-1 enzymatic activity, biliverdin and carbon monoxide (CO), actively contribute to this protection, suggesting that exploitation of these cellular systems may offer new therapeutic avenues in a variety of diseases. Starting from a CO-releasing compound and a chemical scaffold exhibiting electrophilic characteristics (esters of fumaric acid), we report the synthesis of hybrid molecules that simultaneously activate Nrf2 and liberate CO. These hybrid compounds, which we termed "HYCOs", release CO to myoglobin and activate the CO-sensitive fluorescent probe COP-1, while also potently inducing nuclear accumulation of Nrf2 and HO-1 expression and activity in different cell types. Thus, we provide here the first example of a new class of pharmacologically active molecules that target the HO-1 pathway by combining an Nrf2 activator coordinated to a CO-releasing group.

Targeting PHGDH reverses the immunosuppressive phenotype of tumor-associated macrophages through α-ketoglutarate and mTORC1 signaling.

Phosphoglycerate dehydrogenase (PHGDH) has emerged as a crucial factor in macromolecule synthesis, neutralizing oxidative stress, and regulating methylation reactions in cancer cells, lymphocytes, and endothelial cells. However, the role of PHGDH in tumor-associated macrophages (TAMs) is poorly understood. Here, we found that the T helper 2 (Th2) cytokine interleukin-4 and tumor-conditioned media upregulate the expression of PHGDH in macrophages and promote immunosuppressive M2 macrophage activation and proliferation. Loss of PHGDH disrupts cellular metabolism and mitochondrial respiration, which are essential for immunosuppressive macrophages. Mechanistically, PHGDH-mediated serine biosynthesis promotes α-ketoglutarate production, which activates mTORC1 signaling and contributes to the maintenance of an M2-like macrophage phenotype in the tumor microenvironment. Genetic ablation of PHGDH in macrophages from tumor-bearing mice results in attenuated tumor growth, reduced TAM infiltration, a phenotypic shift of M2-like TAMs toward an M1-like phenotype, downregulated PD-L1 expression and enhanced antitumor T-cell immunity. Our study provides a strong basis for further exploration of PHGDH as a potential target to counteract TAM-mediated immunosuppression and hinder tumor progression.

Metabolic support by macrophages sustains colonic epithelial homeostasis.

The intestinal epithelium has a high turnover rate and constantly renews itself through proliferation of intestinal crypt cells, which depends on insufficiently characterized signals from the microenvironment. Here, we showed that colonic macrophages were located directly adjacent to epithelial crypt cells in mice, where they metabolically supported epithelial cell proliferation in an mTORC1-dependent manner. Specifically, deletion of tuberous sclerosis complex 2 (Tsc2) in macrophages activated mTORC1 signaling that protected against colitis-induced intestinal damage and induced the synthesis of the polyamines spermidine and spermine. Epithelial cells ingested these polyamines and rewired their cellular metabolism to optimize proliferation and defense. Notably, spermine directly stimulated proliferation of colon epithelial cells and colon organoids. Genetic interference with polyamine production in macrophages altered global polyamine levels in the colon and modified epithelial cell proliferation. Our results suggest that macrophages act as "commensals" that provide metabolic support to promote efficient self-renewal of the colon epithelium.

Role of the distal hydrogen-bonding network in regulating oxygen affinity in the truncated hemoglobin III from Campylobacter jejuni.

Oxygen affinity in heme-containing proteins is determined by a number of factors, such as the nature and conformation of the distal residues that stabilize the heme bound-oxygen via hydrogen-bonding interactions. The truncated hemoglobin III from Campylobacter jejuni (Ctb) contains three potential hydrogen-bond donors in the distal site: TyrB10, TrpG8, and HisE7. Previous studies suggested that Ctb exhibits an extremely slow oxygen dissociation rate due to an interlaced hydrogen-bonding network involving the three distal residues. Here we have studied the structural and kinetic properties of the G8(WF) mutant of Ctb and employed state-of-the-art computer simulation methods to investigate the properties of the O(2) adduct of the G8(WF) mutant, with respect to those of the wild-type protein and the previously studied E7(HL) and/or B10(YF) mutants. Our data indicate that the unique oxygen binding properties of Ctb are determined by the interplay of hydrogen-bonding interactions between the heme-bound ligand and the surrounding TyrB10, TrpG8, and HisE7 residues.

The single-domain globin from the pathogenic bacterium Campylobacter jejuni: novel D-helix conformation, proximal hydrogen bonding that influences ligand binding, and peroxidase-like redox properties.

The food-borne pathogen Campylobacter jejuni possesses a single-domain globin (Cgb) whose role in detoxifying nitric oxide has been unequivocally demonstrated through genetic and molecular approaches. The x-ray structure of cyanide-bound Cgb has been solved to a resolution of 1.35 A. The overall fold is a classic three-on-three alpha-helical globin fold, similar to that of myoglobin and Vgb from Vitreoscilla stercoraria. However, the D region (defined according to the standard globin fold nomenclature) of Cgb adopts a highly ordered alpha-helical conformation unlike any previously characterized members of this globin family, and the GlnE7 residue has an unexpected role in modulating the interaction between the ligand and the TyrB10 residue. The proximal hydrogen bonding network in Cgb demonstrates that the heme cofactor is ligated by an imidazolate, a characteristic of peroxidase-like proteins. Mutation of either proximal hydrogen-bonding residue (GluH23 or TyrG5) results in the loss of the high frequency nu(Fe-His) stretching mode (251 cm(-1)), indicating that both residues are important for maintaining the anionic character of the proximal histidine ligand. Cyanide binding kinetics for these proximal mutants demonstrate for the first time that proximal hydrogen bonding in globins can modulate ligand binding kinetics at the distal site. A low redox midpoint for the ferrous/ferric couple (-134 mV versus normal hydrogen electrode at pH 7) is consistent with the peroxidase-like character of the Cgb active site. These data provide a new insight into the mechanism via which Campylobacter may survive host-derived nitrosative stress.

Activation of Downstream mTORC1 Target Ribosomal Protein S6 Kinase (S6K) Can Be Found in a Subgroup of Dutch Patients with Granulomatous Pulmonary Disease.

Mechanistic target of rapamycin complex 1 (mTORC1) has been linked to different diseases. The mTORC1 signaling pathway is suggested to play a role in the granuloma formation of sarcoidosis. Recent studies demonstrated conflicting data on mTORC1 activation in patients with sarcoidosis by measuring activation of its downstream target S6 kinase (S6K) with either 33% or 100% of patients. Therefore, the aim of our study was to reevaluate the percentage of S6K activation in sarcoidosis patients in a Dutch cohort. To investigate whether this activation is specific for sarcoid granulomas, we also included Dutch patients with other granulomatous diseases of the lung. The activation of the S6K signaling pathway was evaluated by immunohistochemical staining of its downstream effector phospho-S6 in tissue sections. Active S6K signaling was detected in 32 (43%) of the sarcoidosis patients. Twelve (31%) of the patients with another granulomatous disorder also showed activated S6K signaling, demonstrating that the mTORC1 pathway may be activated in a range for different granulomatous diseases (p = 0.628). Activation of S6K can only be found in a subgroup of patients with sarcoidosis, as well as in patients with other granulomatous pulmonary diseases, such as hypersensitivity pneumonitis or vasculitis. No association between different clinical phenotypes and S6K activation can be found in sarcoidosis.

Inverse Data-Driven Modeling and Multiomics Analysis Reveals Phgdh as a Metabolic Checkpoint of Macrophage Polarization and Proliferation.

Mechanistic or mammalian target of rapamycin complex 1 (mTORC1) is an important regulator of effector functions, proliferation, and cellular metabolism in macrophages. The biochemical processes that are controlled by mTORC1 are still being defined. Here, we demonstrate that integrative multiomics in conjunction with a data-driven inverse modeling approach, termed COVRECON, identifies a biochemical node that influences overall metabolic profiles and reactions of mTORC1-dependent macrophage metabolism. Using a combined approach of metabolomics, proteomics, mRNA expression analysis, and enzymatic activity measurements, we demonstrate that Tsc2, a negative regulator of mTORC1 signaling, critically influences the cellular activity of macrophages by regulating the enzyme phosphoglycerate dehydrogenase (Phgdh) in an mTORC1-dependent manner. More generally, while lipopolysaccharide (LPS)-stimulated macrophages repress Phgdh activity, IL-4-stimulated macrophages increase the activity of the enzyme required for the expression of key anti-inflammatory molecules and macrophage proliferation. Thus, we identify Phgdh as a metabolic checkpoint of M2 macrophages.

Metabolic Programming of Macrophages: Implications in the Pathogenesis of Granulomatous Disease.

Metabolic reprogramming is rapidly gaining appreciation in the etiology of immune cell dysfunction in a variety of diseases. Tuberculosis, schistosomiasis, and sarcoidosis represent an important class of diseases characterized by the formation of granulomas, where macrophages are causatively implicated in disease pathogenesis. Recent studies support the incidence of macrophage metabolic reprogramming in granulomas of both infectious and non-infectious origin. These publications identify the mechanistic target of rapamycin (mTOR), as well as the major regulators of lipid metabolism and cellular energy balance, peroxisome proliferator receptor gamma (PPAR-γ) and adenosine monophosphate-activated protein kinase (AMPK), respectively, as key players in the pathological progression of granulomas. In this review, we present a comprehensive breakdown of emerging research on the link between macrophage cell metabolism and granulomas of different etiology, and how parallels can be drawn between different forms of granulomatous disease. In particular, we discuss the role of PPAR-γ signaling and lipid metabolism, which are currently the best-represented metabolic pathways in this context, and we highlight dysregulated lipid metabolism as a common denominator in granulomatous disease progression. This review therefore aims to highlight metabolic mechanisms of granuloma immune cell fate and open up research questions for the identification of potential therapeutic targets in the future.

HYCO-3, a dual CO-releaser/Nrf2 activator, reduces tissue inflammation in mice challenged with lipopolysaccharide.

Oxidative stress and inflammation are predominant features of several chronic diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a major arbiter in counteracting these insults via up-regulation of several defensive proteins, including heme oxygenase-1 (HO-1). HO-1-derived carbon monoxide (CO) exhibits anti-inflammatory actions and can be delivered to tissues by CO-releasing agents. In this study we assessed the pharmacological and anti-inflammatory properties of HYCO-3, a dual activity compound obtained by conjugating analogues of the CO-releasing molecule CORM-401 and dimethyl fumarate (DMF), an immunomodulatory drug known to activate Nrf2. HYCO-3 induced Nrf2-dependent genes and delivered CO to cells in vitro and tissues in vivo, confirming that the two expected pharmacological properties of this agent are achieved. In mice challenged with lipopolysaccharide, orally administered HYCO-3 reduced the mRNA levels of pro-inflammatory markers (TNF-α, IL-1ß and IL-6) while increasing the expression of the anti-inflammatory genes ARG1 and IL-10 in brain, liver, lung and heart. In contrast, DMF or CORM-401 alone or their combination decreased the expression of pro-inflammatory genes but had limited influence on anti-inflammatory markers. Furthermore, HYCO-3 diminished TNF-α and IL-1ß in brain and liver but not in lung and heart of Nrf2-/- mice, indicating that the CO-releasing part of this hybrid contributes to reduction of pro-inflammation and that this effect is organ-specific. These data demonstrate that the dual activity of HYCO-3 results in enhanced efficacy compared to the parent compounds indicating the potential exploitation of hybrid compounds in the development of effective anti-inflammatory therapies.

Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus.

Microglia fulfill important immunological functions in the brain by responding to pathological stresses and modulating their activities according to pro- or anti-inflammatory stimuli. Recent evidence indicates that changes in metabolism accompany the switch in microglia activation state, favoring glycolysis over oxidative phosphorylation when cells exhibit a pro-inflammatory phenotype. Carbon monoxide (CO), a byproduct of heme breakdown by heme oxygenase, exerts anti-inflammatory action and affects mitochondrial function in cells and tissues. In the present study, we analyzed the metabolic profile of BV2 and primary mouse microglia exposed to the CO-releasing molecules CORM-401 and CORM-A1 and investigated whether CO affects the metabolic adaptation of cells to the inflammatory stimulus lipopolysaccharide (LPS). Microglia respiration and glycolysis were measured using an Extracellular Flux Analyzer to provide a real-time bioenergetic assessment, and biochemical parameters were evaluated to define the metabolic status of the cells under normal or inflammatory conditions. We show that CO prevents LPS-induced depression of microglia respiration and reduction in ATP levels while altering the early expression of inflammatory markers, suggesting the metabolic changes induced by CO are associated with control of inflammation. CO alone affects microglia respiration depending on the concentration, as low levels increase oxygen consumption while higher amounts inhibit respiration. Increased oxygen consumption was attributed to an uncoupling activity observed in cells, at the molecular level (respiratory complex activities) and during challenge with LPS. Thus, application of CO is a potential countermeasure to reverse the metabolic changes that occur during microglia inflammation and in turn modulate their inflammatory profile.

Diverse Nrf2 Activators Coordinated to Cobalt Carbonyls Induce Heme Oxygenase-1 and Release Carbon Monoxide in Vitro and in Vivo.

The Nrf2/heme oxygenase-1 (HO-1) axis affords significant protection against oxidative stress and cellular damage. We synthesized a series of cobalt-based hybrid molecules (HYCOs) that combine an Nrf2 inducer with a releaser of carbon monoxide (CO), an anti-inflammatory product of HO-1. Two HYCOs markedly increased Nrf2/HO-1 expression, liberated CO and exerted anti-inflammatory activity in vitro. HYCOs also up-regulated tissue HO-1 and delivered CO in blood after administration in vivo, supporting their potential use against inflammatory conditions.

Vascular and angiogenic activities of CORM-401, an oxidant-sensitive CO-releasing molecule.

Carbon monoxide (CO) is generated by heme oxygenase-1 (HO-1) and displays important signaling, anti-apoptotic and anti-inflammatory activities, indicating that pharmacological agents mimicking its action may have therapeutic benefit. This study examined the biochemical and pharmacological properties of CORM-401, a recently described CO-releasing molecule containing manganese as a metal center. We used in vitro approaches, ex-vivo rat aortic rings and the EA.hy926 endothelial cell line in culture to address how CORM-401 releases CO and whether the compound modulates vascular tone and pro-angiogenic activities, respectively. We found that CORM-401 released up to three CO/mole of compound depending on the concentration of the acceptor myoglobin. Oxidants such as H2O2, tert-butyl hydroperoxide or hypochlorous acid increased the CO liberated by CORM-401. CORM-401 also relaxed pre-contracted aortic rings and vasorelaxation was enhanced in combination with H2O2. Consistent with the release of multiple CO molecules, CORM-401-induced vasodilation was three times higher than that elicited by CORM-A1, which exhibits a similar half-life to CORM-401 but liberates only one CO/mole of compound. Furthermore, endothelial cells exposed to CORM-401 accumulated CO intracellularly, accelerated migration in vitro and increased VEGF and IL-8 levels. Studies using pharmacological inhibitors revealed HO-1 and p38 MAP kinase as two independent and parallel mechanisms involved in stimulating migration. We conclude that the ability of CORM-401 to release multiple CO, its sensitivity to oxidants which increase CO release, and its vascular and pro-angiogenic properties highlight new advances in the design of CO-releasing molecules that can be tailored for the treatment of inflammatory and oxidative stress-mediated pathologies.

Analysis of transcript changes in a heme-deficient mutant of Escherichia coli in response to CORM-3 [Ru(CO)3Cl(glycinate)].

This article describes in extended detail the methodology applied for acquisition of transcriptomic data, and subsequent statistical data modelling, published by Wilson et al. (2015) in a study of the effects of carbon monoxide-releasing molecule-3 (CORM-3 [Ru(CO)3Cl(glycinate)]) on heme-deficient bacteria. The objective was to identify non-heme targets of CORM action. Carbon monoxide (CO) interacts with heme-containing proteins, in particular respiratory cytochromes; however, CORMs have been shown to elicit multifaceted effects in bacteria, suggesting that the compounds may have additional targets. We therefore sought to elucidate the activity of CORM-3, the first water-soluble CORM and one of the most characterised CORMs to date, in bacteria devoid of heme synthesis. Importantly, we also tested inactive CORM-3 (iCORM-3), a ruthenium co-ligand fragment that does not release CO, in order to differentiate between CO- and compound-related effects. A well-established hemA mutant of Escherichia coli was used for the study and, for comparison, parallel experiments were performed on the corresponding wild-type strain. Global transcriptomic changes induced by CORM-3 and iCORM-3 were evaluated using a Two-Color Microarray-Based Prokaryote Analysis (FairPlay III Labeling) by Agilent Technologies (Inc. 2009). Data acquisition was carried out using Agilent Feature Extraction software (v6.5) and data normalisation, as well as information about gene products and their function was obtained from GeneSpring GX v7.3 (Agilent Technologies). Functional category lists were created using KEGG (Kyoto Encyclopedia of Genes and Genomes). Relevant regulatory proteins for each gene were identified, where available, using regulonDB and EcoCyc (World Wide Web). Statistical data modelling was performed on the gene expression data to infer transcription factor activities. The transcriptomic data can be accessed through NCBI's Gene Expression Omnibus (GEO): series accession number GSE55097 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55097).

CO-Releasing Molecules Have Nonheme Targets in Bacteria: Transcriptomic, Mathematical Modeling and Biochemical Analyses of CORM-3 [Ru(CO)3Cl(glycinate)] Actions on a Heme-Deficient Mutant of Escherichia coli.

AIMS: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy. Hemes are generally considered the prime targets of CO and CORMs, so we tested this hypothesis using heme-deficient bacteria, applying cellular, transcriptomic, and biochemical tools. RESULTS: CORM-3 [Ru(CO)3Cl(glycinate)] readily penetrated Escherichia coli hemA bacteria and was inhibitory to these and Lactococcus lactis, even though they lack all detectable hemes. Transcriptomic analyses, coupled with mathematical modeling of transcription factor activities, revealed that the response to CORM-3 in hemA bacteria is multifaceted but characterized by markedly elevated expression of iron acquisition and utilization mechanisms, global stress responses, and zinc management processes. Cell membranes are disturbed by CORM-3. INNOVATION: This work has demonstrated for the first time that CORM-3 (and to a lesser extent its inactivated counterpart) has multiple cellular targets other than hemes. A full understanding of the actions of CORMs is vital to understand their toxic effects. CONCLUSION: This work has furthered our understanding of the key targets of CORM-3 in bacteria and raises the possibility that the widely reported antimicrobial effects cannot be attributed to classical biochemical targets of CO. This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

Ru(CO)3Cl(Glycinate) (CORM-3): a carbon monoxide-releasing molecule with broad-spectrum antimicrobial and photosensitive activities against respiration and cation transport in Escherichia coli.

AIMS: Carbon monoxide (CO) delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas. The metal carbonyl Ru(CO)3Cl(glycinate) (CORM-3) is a novel, potent antimicrobial agent. Here, we established its mode of action. RESULTS: CORM-3 inhibits respiration in several bacterial and yeast pathogens. In anoxic Escherichia coli suspensions, CORM-3 first stimulates, then inhibits respiration, but much higher concentrations of CORM-3 than of a classic protonophore are required for stimulation. Proton translocation measurements (H(+)/O quotients, i.e., H(+) extrusion on pulsing anaerobic cells with O2) show that respiratory stimulation cannot be attributed to true "uncoupling," that is, dissipation of the protonmotive force, or to direct stimulation of oxidase activity. Our data are consistent with CORM-3 facilitating the electrogenic transmembrane movement of K(+) (or Na(+)), causing a stimulation of respiration and H(+) pumping to compensate for the transient drop in membrane potential (ΔΨ). The effects on respiration are not mimicked by CO gas or control Ru compounds that do not release CO. Inhibition of respiration and loss of bacterial viability elicited by CORM-3 are reversible by white light, unambiguously identifying heme-containing oxidase(s) as target(s). INNOVATION: This is the most complete study to date of the antimicrobial action of a CO-RM. Noteworthy are the demonstration of respiratory stimulation, electrogenic ion transport, and photosensitive activity, establishing terminal oxidases and ion transport as primary targets. CONCLUSION: CORM-3 has multifaceted effects: increased membrane permeability, inhibition of terminal oxidases, and perhaps other unidentified mechanisms underlie its effectiveness in tackling microbial pathogenesis.

Antibacterial effects of carbon monoxide.

Carbon monoxide (CO) is a colourless and odourless gas that has long been considered as a potent respiratory poison. Recent advances have demonstrated its production by haem oxygenases in both mammals and microbes, and it has roles as a gasotransmitter in higher organisms. This review concentrates on the application of CO, via carbon monoxide-releasing molecules (CO-RMs), as an anti-bacterial agent. Currently, the scope of literature on the effects of CO on bacteria is small, and we have included discussions on the production of CO by bacteria via haem oxygenase enzymes, the use of CO as an energy source, and existing knowledge on CO sensors in bacteria. CO is known to target haem proteins and is an effective inhibitor of respiration, even when provided at concentrations much higher than prevailing oxygen. We review here data suggesting that CO-RMs are more effective inhibitors of respiration than is CO gas, perhaps due to the ability of CO-RMs to deliver CO selectively to intracellular targets. We also consider the recently reported transcriptomic consequences of CO-RM treatment of Escherichia coli, revealing a myriad of unexpected targets for CO and potential CO sensors. Finally, we consider the use of CO and CO-RMs as anti-bacterial agents in vivo, and the future prospects for this gaseous molecule.

Carbon monoxide in biology and microbiology: surprising roles for the "Detroit perfume".

Carbon monoxide (CO) is a colorless, odorless gas with a reputation for being an anthropogenic poison; there is extensive documentation of the modes of human exposure, toxicokinetics, and health effects. However, CO is also generated endogenously by heme oxygenases (HOs) in mammals and microbes, and its extraordinary biological activities are now recognized and increasingly utilized in medicine and physiology. This review introduces recent advances in CO biology and chemistry and illustrates the exciting possibilities that exist for a deeper understanding of its biological consequences. However, the microbiological literature is scant and is currently restricted to: 1) CO-metabolizing bacteria, CO oxidation by CO dehydrogenase (CODH) and the CO-sensing mechanisms that enable CO oxidation; 2) the use of CO as a heme ligand in microbial biochemistry; and 3) very limited information on how microbes respond to CO toxicity. We demonstrate how our horizons in CO biology have been extended by intense research activity in recent years in mammalian and human physiology and biochemistry. CO is one of several "new" small gas molecules that are increasingly recognized for their profound and often beneficial biological activities, the others being nitric oxide (NO) and hydrogen sulfide (H2S). The chemistry of CO and other heme ligands (oxygen, NO, H2S and cyanide) and the implications for biological interactions are briefly presented. An important advance in recent years has been the development of CO-releasing molecules (CO-RMs) for aiding experimental administration of CO as an alternative to the use of CO gas. The chemical principles of CO-RM design and mechanisms of CO release from CO-RMs (dissociation, association, reduction and oxidation, photolysis, and acidification) are reviewed and we present a survey of the most commonly used CO-RMs. Amongst the most important new applications of CO in mammalian physiology and medicine are its vasoactive properties and the therapeutic potentials of CO-RMs in vascular disease, anti-inflammatory effects, CO-mediated cell signaling in apoptosis, applications in organ preservation, and the effects of CO on mitochondrial function. The very limited literature on microbial growth responses to CO and CO-RMs in vitro, and the transcriptomic and physiological consequences of microbial exposure to CO and CO-RMs are reviewed. There is current interest in CO and CO-RMs as antimicrobial agents, particularly in the control of bacterial infections. Future prospects are suggested and unanswered questions posed.