Publications by authors named "Eduardo Henrique Silva Sousa"

22 Publications

  • Page 1 of 1

Synthesis and potential vasorelaxant effect of a novel ruthenium-based nitro complex.

J Inorg Biochem 2022 Mar 26;228:111666. Epub 2021 Nov 26.

Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Coronel Nunes de Melo St., 1127, 60.430-275 Fortaleza, CE, Brazil; Drug Research and Development Center (NPDM), Federal University of Ceará, Coronel Nunes de Melo St., 1000, 60.430-275 Fortaleza, CE, Brazil.

This study aimed to investigate the synthesis and potential vasodilator effect of a novel ruthenium complex, cis-[Ru(bpy)(2-MIM)(NO)]PF (bpy = 2,2'-bipyridine and 2-MIM = 2-methylimidazole) (FOR711A), containing an imidazole derivative via an in silico molecular docking model using β1 H-NOX (Heme-nitric oxide/oxygen binding) domain proteins of reduced and oxidized soluble guanylate cyclase (sGC). In addition, pharmacokinetic properties in the human organism were predicted through computational simulations and the potential for acute irritation of FOR711A was also investigated in vitro using the hen's egg chorioallantoic membrane (HET-CAM). FOR711A interacted with sites of the β1 H-NOX domain of reduced and oxidized sGC, demonstrating shorter bond distances to several residues and negative values of total energy. The predictive study revealed molar refractivity (RM): 127.65; Log Po/w = 1.29; topological polar surface area (TPSA): 86.26 Å; molar mass (MM) = 541.55 g/mol; low solubility, high unsaturation index, high gastrointestinal absorption; toxicity class 4; failure to cross the blood-brain barrier and to react with cytochrome P450 (CYP) enzymes CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4. After the HET-CAM assay, the FOR711A complex was classified as non-irritant (N.I.) and its vasodilator effect was confirmed through greater evidence of blood vessels after the administration and ending of the observation period of 5 min. These results suggest that FOR711A presented a potential stimulator/activator effect of sGC via NO/sGC/cGMP. However, results indicate it needs a vehicle for oral administration.
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http://dx.doi.org/10.1016/j.jinorgbio.2021.111666DOI Listing
March 2022

The biofilm inhibition activity of a NO donor nanosilica with enhanced antibiotics action.

Int J Pharm 2021 Dec 20;610:121220. Epub 2021 Oct 20.

Departamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, Fortaleza, CE 60440-900, Brazil. Electronic address:

Nitric oxide (NO) has emerged as a promising antibacterial agent, where NO donor compounds have been explored. Here, we investigated the role of a silica nanoparticle containing nitroprusside (MPSi-NP) as a NO donor agent against methicillin-sensitive (ATCC 25,923 and ATCC 12228) and methicillin-resistant (ATCC 700,698 and ATCC 35984) Staphylococcus strains. Biofilm inhibition was studied along with antibiotic activity in combination with standard antibiotics (ampicillin and tetracycline). MPSi-NP exhibited thermal release of 63% of NO within 24 h, while free nitroprusside released only 18% during a dialysis assay, indicating an assisted release of NO mediated by the nanoparticles. This nanomaterial showed only a moderate activity in blocking biofilm production, but exhibited a significant decrease in the number of viable bacterial cells (over 600-fold for Staphylococcus aureus ATCC 700,698 and Staphylococcus epidermidis ATCC 35984). Remarkably, even using MPSi-NP at concentrations below any antibacterial action, its combination with ampicillin promoted a significant decrease in MIC for resistant strains of S. aureus ATCC 700,698 (2-fold) and S. epidermidis ATCC 35,984 (4-fold). A carbopol-based gel formulation with MPSi-NP (0.5% w/w) was prepared and showed a zone of inhibition of 7.7 ± 0.6 mm for S. epidermidis ATCC 35984. Topical use of MPSi-NP in combination with antibiotics might be a manageable strategy to prevent and eventually treat complicated resistant bacterial infections.
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http://dx.doi.org/10.1016/j.ijpharm.2021.121220DOI Listing
December 2021

Further Insights into the Oxidative Pathway of Thiocarbonyl-Type Antitubercular Prodrugs: Ethionamide, Thioacetazone, and Isoxyl.

Chem Res Toxicol 2021 08 28;34(8):1879-1889. Epub 2021 Jul 28.

CNRS, Laboratoire de Chimie de Coordination, LCC, UPR 8241, 205 Route de Narbonne, BP 44099, F-31077 Toulouse, Cedex 4, France.

A chemical activation study of the thiocarbonyl-type antitubercular prodrugs, ethionamide (ETH), thioacetazone (TAZ), and isoxyl (ISO), was performed. Biomimetic oxidation of ethionamide using HO (1 equiv) led to ETH-SO as the only stable -oxide compound, which was found to occur in solution in the preferential form of a sulfine (ETH═S═O vs the sulfenic acid tautomer ETH-S-OH), as previously observed in the crystal state. It was also demonstrated that ETH-SO is capable of reacting with amines, as the putative sulfinic derivative (ETH-SOH) was supposed to do. Unlike ETH, oxidation of TAZ did not allow observation of the mono-oxygenated species (TAZ-SO), leading directly to the more stable sulfinic acid derivative (TAZ-SOH), which can then lose a SOH group after further oxidation or when placed in a basic medium. It was also noticed that the unstable TAZ-SO intermediate can lead to the carbodiimide derivative as another electrophilic species. It is suggested that TAZ-SOH, TAZ-SOH, and the carbodiimide compound can also react with NH-containing nucleophilic species, and therefore be involved in toxic effects. Finally, ISO showed a very complex reactivity, here assigned to the coexistence of two mono-oxygenated structures, the sulfine and sulfenic acid tautomers. The mono- and dioxygenated derivatives of ISO are also highly unstable, leading to a panel of multiple metabolites, which are still reactive and likely contribute to the toxicity of this prodrug.
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http://dx.doi.org/10.1021/acs.chemrestox.1c00164DOI Listing
August 2021

Anti-asthmatic effect of nitric oxide metallo-donor FOR811A [cis-[Ru(bpy)2(2-MIM)(NO)](PF6)3] in the respiratory mechanics of Swiss mice.

PLoS One 2021 12;16(3):e0248394. Epub 2021 Mar 12.

Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, CE, Brazil.

We aimed at evaluating the anti-asthmatic effect of cis-[Ru(bpy)2(2-MIM)(NO)](PF6)3 (FOR811A), a nitrosyl-ruthenium compound, in a murine model of allergic asthma. The anti-asthmatic effects were analyzed by measuring the mechanical lung and morphometrical parameters in female Swiss mice allocated in the following groups: untreated control (Ctl+Sal) and control treated with FOR811A (Ctl+FOR), along asthmatic groups untreated (Ast+Sal) and treated with FOR811A (Ast+FOR). The drug-protein interaction was evaluated by in-silico assay using molecular docking. The results showed that the use of FOR811A in experimental asthma (Ast+FOR) decreased the pressure-volume curve, hysteresis, tissue elastance, tissue resistance, and airway resistance, similar to the control groups (Ctl+Sal; Ctl+FOR). However, it differed from the untreated asthmatic group (Ast+Sal, p<0.05), indicating that FOR811A corrected the lung parenchyma and relaxed the smooth muscles of the bronchi. Similar to control groups (Ctl+Sal; Ctl+FOR), FOR811A increased the inspiratory capacity and static compliance in asthmatic animals (Ast+Sal, p<0.05), showing that this metallodrug improved the capacity of inspiration during asthma. The morphometric parameters showed that FOR811A decreased the alveolar collapse and kept the bronchoconstriction during asthma. Beyond that, the molecular docking using FOR811A showed a strong interaction in the distal portion of the heme group of the soluble guanylate cyclase, particularly with cysteine residue (Cys141). In summary, FOR811A relaxed bronchial smooth muscles and improved respiratory mechanics during asthma, providing a protective effect and promising use for the development of an anti-asthmatic drug.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248394PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954307PMC
October 2021

Pentacyanoferrate(II) complex of pyridine-4- and pyrazine-2-hydroxamic acid as source of HNO: investigation of anti-tubercular and vasodilation activities.

J Biol Inorg Chem 2020 09 29;25(6):887-901. Epub 2020 Jul 29.

CNRS, Laboratoire de Chimie de Coordination, LCC, UPR 8241, 205 Route de Narbonne, BP 44099, 31077, Cedex 4 Toulouse, France.

A pharmacophore design approach, based on the coordination chemistry of an intimate molecular hybrid of active metabolites of pro-drugs, known to release active species upon enzymatic oxidative activation, is devised. This is exemplified by combining two anti-mycobacterial drugs: pyrazinamide (first line) and delamanid (third line) whose active metabolites are pyrazinoic acid (PyzCOOH) and likely nitroxyl (HNO (or NO)), respectively. Aiming to generate those active species, a hybrid compound was envisaged by coordination of pyrazine-2-hydroxamic acid (PyzCONHOH) with a Na[Fe(CN)] moiety. The corresponding pentacyanoferrate(II) complex Na[Fe(CN)(PyzCONHO)] was synthesized and characterized by several spectroscopic techniques, cyclic voltammetry, and DFT calculations. Chemical oxidation of this complex with HO was shown to induce the release of the metabolite PyzCOOH, without the need of the Mycobacterium tuberculosis (Mtb) pyrazinamidase enzyme (PncA). Control experiments show that both HO- and N-coordinated pyrazine Fe species are required, ruling out a direct hydrolysis of the hydroxamic acid or an alternative oxidative route through chelation of a metal center by a hydroxamic group. The release of HNO was observed using EPR spectroscopy in the presence of a spin trapping agent. The devised iron metal complex of pyrazine-2-hydroxamic acid was found inactive against an actively growing/non-resistant Mtb strain; however, it showed a strong dose-dependent and reversible vasodilatory activity with mostly lesser toxic effects than the reference drug sodium nitroprussiate, unveiling thus a potential indication for acute or chronic cardiovascular pathology. This is a priori a further indirect evidence of HNO release from this metal complex, standing as a possible pharmacophore model for an alternative vasodilator drug.
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http://dx.doi.org/10.1007/s00775-020-01805-zDOI Listing
September 2020

A divergent mode of activation of a nitrosyl iron complex with unusual antiangiogenic activity.

J Inorg Biochem 2020 09 20;210:111133. Epub 2020 Jun 20.

Departamento de Química Orgânica e Inorgânica, Grupo de Bioinorgânica, Universidade Federal do Ceará-UFC, P.O Box 6021, Fortaleza, CE CEP 60440-900, Brazil. Electronic address:

Nitric oxide (NO) and nitroxyl (HNO) have gained broad attention due to their roles in several physiological and pathophysiological processes. Remarkably, these sibling species can exhibit opposing effects including the promotion of angiogenic activity by NO compared to HNO, which blocks neovascularization. While many NO donors have been developed over the years, interest in HNO has led to the recent emergence of new donors. However, in both cases there is an expressive lack of iron-based compounds. Herein, we explored the novel chemical reactivity and stability of the trans-[Fe(cyclam)(NO)Cl]Cl (cyclam = 1,4,8,11-tetraazacyclotetradecane) complex. Interestingly, the half-life (t) for NO release was 1.8 min upon light irradiation, vs 5.4 h upon thermal activation at 37 °C. Importantly, spectroscopic evidence supported the generation of HNO rather than NO induced by glutathione. Moreover, we observed significant inhibition of NO donor- or hypoxia-induced HIF-1α (hypoxia-inducible factor 1α) accumulation in breast cancer cells, as well as reduced vascular tube formation by endothelial cells pretreated with the trans-[Fe(cyclam)(NO)Cl]Cl complex. Together, these studies provide the first example of an iron-nitrosyl complex with anti-angiogenic activity as well as the potential dual activity of this compound as a NO/HNO releasing agent, which warrants further pharmacological investigation.
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http://dx.doi.org/10.1016/j.jinorgbio.2020.111133DOI Listing
September 2020

Antimicrobial activity and antibiotic synergy of a biphosphinic ruthenium complex against clinically relevant bacteria.

Biofouling 2020 04 25;36(4):442-454. Epub 2020 May 25.

Laboratório Integrado de Biomoléculas, Departamento de Patologia e Medicina Legal, Universidade Federal do Ceará, Fortaleza, CE, Brasil.

The aim of this study was to investigate the antibacterial activity, antibiotic-associated synergy, and anti-biofilm activity of the ruthenium complex, -[RuCl (dppb) (bqdi)] (RuNN). RuNN exhibited antimicrobial activity against Gram-positive bacteria with minimum inhibitory concentration (MIC) values ranging from 15.6 to 62.5 µg ml and minimum bactericidal concentration (MBC) values ranging from 62.5 to 125 µg ml. A synergistic effect against spp. was observed when RuNN was combined with ampicillin, and the range of associated fractional inhibitory concentration index (FICI) values was 0.187 to 0.312. A time-kill curve indicated the bactericidal activity of RuNN in the first 1-5 h. In general, RuNN inhibited biofilm formation and disrupted mature biofilms. Furthermore, RuNN altered the cellular morphology of biofilms. Further, RuNN did not cause hemolysis of erythrocytes. The results of this study provide evidence that RuNN is a novel therapeutic candidate to treat bacterial infections caused by biofilms.
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http://dx.doi.org/10.1080/08927014.2020.1771317DOI Listing
April 2020

Antihypertensive potential of cis-[Ru(bpy)(ImN)(NO)], a ruthenium-based nitric oxide donor.

Res Vet Sci 2020 Jun 7;130:153-160. Epub 2020 Mar 7.

Superior Institute of Biomedical Sciences, Ceará State University, Fortaleza, CE, Brazil. Electronic address:

The aim of this study was to investigate the antihypertensive properties of cis-[Ru(bpy)ImN(NO)] (FOR0811) in normotensive and in N-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats. Vasorelaxant effects were analyzed by performing concentration response curve to FOR0811 in rat aortic rings in the absence or presence of 1H-[1,2,4]-oxadiazolo-[4,3,-a]quinoxalin-1-one (ODQ), L-cysteine or hydroxocobalamin. Normotensive and L-NAME-hypertensive rats were treated with FOR0811 and the effects in blood pressure and heart rate variability in the frequency domain (HRV) were followed. FOR0811 induced relaxation in rat aortic rings. Neither endothelium removal nor L-cysteine altered the FOR0811 effects. However, the incubation with ODQ and hydroxocobalamin completely blunted FOR0811 effects. FOR0811 administered intravenously by bolus infusion (0.01-1 mg/bolus) or chronically by using subcutaneous implanted osmotic pumps significantly reduced the mean arterial blood pressure. The effect was long lasting and did not induce reflex tachycardia. FOR0811 prevented both LF and VLF increases in L-NAME hypertensive rats and has antihypertensive properties. This new ruthenium complex compound might be a promising nitric oxide donor to treat cardiovascular diseases.
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http://dx.doi.org/10.1016/j.rvsc.2020.03.014DOI Listing
June 2020

Nitro-imidazole-based ruthenium complexes with antioxidant and anti-inflammatory activities.

J Inorg Biochem 2020 05 24;206:111048. Epub 2020 Feb 24.

Department of Organic and Inorganic Chemistry, Universidade Federal do Ceará, PO Box 6021, Fortaleza, Brazil. Electronic address:

Inflammation is a physiological process triggered in response to tissue damage, and involves events related to cell recruitment, cytokines release and reactive oxygen species (ROS) production. Failing to control the process duration lead to chronification and may be associated with the development of various pathologies, including autoimmune diseases and cancer. Considering the pharmacological potential of metal-based compounds, two new ruthenium complexes were synthesized: cis-[Ru(NO)(bpy)(5NIM)]PF (1) and cis-[RuCl(bpy)(MTZ)]PF (2), where bpy = 2,2'-bipyridine, 5NIM = 5-nitroimidazole and MTZ = metronidazole. Both products were characterized by spectroscopic techniques, followed by Density Functional Theory (DFT) calculations in order to support experimental findings. Afterwards, their in vitro cytotoxic, antioxidant and anti-inflammatory activities were investigated. Compounds 1 and 2 presented expressive in vitro antioxidant activity, reducing lipid peroxidation and decreasing intracellular ROS levels with comparable effectiveness to the standard steroidal drug dexamethasone or α-tocopherol. These complexes showed no noticeable cytotoxicity on the tested cancer cell lines. Bactericidal assay against metronidazole-resistant Helicobacter pylori, a microorganism able to disrupt oxidative balance, unraveled compound 1 moderate activity over that strain. Besides this, it was able to inhibit interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) production as well as interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. This latter activity is remarkable, which has not been reported for other ruthenium-based complexes. Altogether, these results suggest cis-[Ru(NO)(bpy)(5NIM)]PF complex has potential pharmacological application as an anti-inflammatory agent that deserve further biological investigation.
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http://dx.doi.org/10.1016/j.jinorgbio.2020.111048DOI Listing
May 2020

In vitro and in vivo leishmanicidal activity of a ruthenium nitrosyl complex against Leishmania (Viannia) braziliensis.

Acta Trop 2019 Apr 25;192:61-65. Epub 2019 Jan 25.

School of Veterinary, Postgraduate Program in Veterinary Sciences, State University of Ceará, Fortaleza, Ceará, Brazil; Department of Pathology and Legal Medicine, School of Medicine, Specialized Medical Mycology Center, Postgraduate Program in Medical Microbiology, Federal University of Ceará, Fortaleza, Ceará, Brazil.

Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania. There are many complications presented by the current treatment, as high toxicity, high cost and parasite resistance, making the development of new therapeutic agents indispensable. The present study aims to evaluate the leishmanicidal potential of ruthenium nitrosyl complex cis-[Ru(bpy)(SO)(NO)](PF) against Leishmania (Viannia) braziliensis. The effect of this metal complex on parasite-host interaction was evaluated by in vitro efficacy test in dermal fibrobast cells in the presence of different concentrations (1, 10, 50 and 100 μM) and by in vivo efficacy tests performed in the presence of two different concentrations of complex (100 μg/kg/day or 300 μg/kg/day) evaluating its effect on the size of the lesion and the number of parasites present in the draining lymph nodes in hamsters. Even at the lowest concentration of 1 μM of ruthenium complex, it was observed a significant decrease of the infected cells, after 24 h exposure in vitro, with total reduction at 50 μM of the ruthenium complex. In the in vivo cutaneous infection model, administration of daily doses of 300 μg/kg/day of complex reduced significantly lesion size by 51% (p < 0.05), with a 99.9% elimination of the parasites found in the lymph nodes (p < 0.001). The results suggest a promising leishmanicidal effect by that ruthenium nitrosyl complex against L. (V.) braziliensis.
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http://dx.doi.org/10.1016/j.actatropica.2019.01.021DOI Listing
April 2019

Synthesis and mechanistic investigation of iron(II) complexes of isoniazid and derivatives as a redox-mediated activation strategy for anti-tuberculosis therapy.

J Inorg Biochem 2018 02 21;179:71-81. Epub 2017 Nov 21.

CNRS, LCC (Laboratoire de Chimie de Coordination), 205, route de Narbonne, BP 44099, F-31077 Toulouse, Cedex 4, France; Université de Toulouse, UPS, INPT, F-31077 Toulouse, Cedex 4, France. Electronic address:

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) represents a major threat to global health. Isoniazid (INH) is a prodrug used in the first-line treatment of tuberculosis. It undergoes oxidation by a catalase-peroxidase KatG, leading to generation of an isonicotinoyl radical that reacts with NAD(H) forming the INH-NADH adduct as the active metabolite. A redox-mediated activation of isoniazid using an iron metal complex was previously proposed as a strategy to overcome isoniazid resistance due to KatG mutations. Here, we have prepared a series of iron metal complexes with isoniazid and analogues, containing alkyl substituents at the hydrazide moiety, and also with pyrazinamide derivatives. These complexes were activated by HO and studied by ESR and LC-MS. For the first time, the formation of the oxidized INH-NAD adduct from the pentacyano(isoniazid)ferrate(II) complex was detected by LC-MS, supporting a redox-mediated activation, for which a mechanistic proposition is reported. ESR data showed all alkylated hydrazides, in contrast to non-substituted hydrazides, only generated alkyl-based radicals. The structural modifications did not improve minimal inhibitory concentration (MIC) against MTB in comparison to isoniazid iron complex, providing support to isonicotinoyl radical formation as a requirement for activity. Nonetheless, the pyrazinoic acid hydrazide iron complex showed redox-mediated activation using HO with generation of a pyrazinoyl radical intermediate and production of pyrazinoic acid, which is in fact the active metabolite of pyrazinamide prodrug. Thereby, this strategy can also unveil new opportunities for activation of this type of drug.
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http://dx.doi.org/10.1016/j.jinorgbio.2017.11.013DOI Listing
February 2018

Aryl-Substituted Ruthenium(II) Complexes: A Strategy for Enhanced Photocleavage and Efficient DNA Binding.

Inorg Chem 2017 Aug 20;56(15):9084-9096. Epub 2017 Jul 20.

Laboratório de Bioinorgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará , Cx. Postal 6021, 60440-900 Fortaleza, Ceará Brazil.

Ruthenium polypyridine complexes have shown promise as agents for photodynamic therapy (PDT) and tools for molecular biology (chromophore-assisted light inactivation). To accomplish these tasks, it is important to have at least target selectivity and great reactive oxygen species (ROS) photogeneration: two properties that are not easily found in the same molecule. To prepare such new agents, we synthesized two new ruthenium complexes that combine an efficient DNA binding moiety (dppz ligand) together with naphthyl-modified (1) and anthracenyl-modified (2) bipyridine as a strong ROS generator bound to a ruthenium complex. The compounds were fully characterized and their photophysical and photochemical properties investigated. Compound 2 showed one of the highest quantum yields for singlet oxygen production ever reported (Φ= 0.96), along with very high DNA binding (log K = 6.78). Such photochemical behavior could be ascribed to the lower triplet state involving the anthracenyl-modified bipyridine, which is associated with easier oxygen quenching. In addition, the compounds exhibited moderate selectivity toward G-quadruplex DNA and binding to the minor groove of DNA, most likely driven by the pendant ligands. Interestingly, they also showed DNA photocleavage activity even upon exposure to a yellow light-emitting diode (LED). Regarding their biological activity, the compounds exhibited an exciting antibacterial action, particularly against Gram-positive bacteria, which was enhanced upon blue LED irradiation. Altogether, these results showed that our strategy succeeded in producing light-triggered DNA binding agents with pharmacological and biotechnological potential.
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http://dx.doi.org/10.1021/acs.inorgchem.7b01108DOI Listing
August 2017

Drug discovery targeting heme-based sensors and their coupled activities.

J Inorg Biochem 2017 02 20;167:12-20. Epub 2016 Nov 20.

Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9038, USA. Electronic address:

Heme-based sensors have emerged during the last 20years as being a large family of proteins that occur in all kingdoms of life. A myriad of biological adaptations are associated with these sensors, which include vasodilation, bacterial virulence, dormancy, chemotaxis, biofilm formation, among others. Due to the key activities regulated by these proteins along with many other systems that use similar output domains, there is a growing interest in developing small molecules as their regulators. Here, we review the development of potential activators and inhibitors for many of these systems, including human soluble guanylate cyclase, c-di-GMP-related enzymes, Mycobacterium tuberculosis DevR/DevS/DosT (differentially expressed in virulent strain response regulator/sensor/dormancysurvival sensorT), the Rev-erb-α and β nuclear receptor, among others. The possible roles of these molecules as biochemical tools, therapeutic agents, and novel antibiotics are critically examined.
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http://dx.doi.org/10.1016/j.jinorgbio.2016.11.022DOI Listing
February 2017

Thiol-Activated HNO Release from a Ruthenium Antiangiogenesis Complex and HIF-1α Inhibition for Cancer Therapy.

ACS Chem Biol 2016 07 31;11(7):2057-65. Epub 2016 May 31.

Department of Chemistry, University of Warwick , Coventry CV4 7AL, United Kingdom.

Metallonitrosyl complexes are promising as nitric oxide (NO) donors for the treatment of cardiovascular, endothelial, and pathogenic diseases, as well as cancer. Recently, the reduced form of NO(-) (protonated as HNO, nitroxyl, azanone, isoelectronic with O2) has also emerged as a candidate for therapeutic applications including treatment of acute heart failure and alcoholism. Here, we show that HNO is a product of the reaction of the Ru(II) complex [Ru(bpy)2(SO3)(NO)](+) (1) with glutathione or N-acetyl-L-cysteine, using met-myoglobin and carboxy-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) as trapping agents. Characteristic absorption spectroscopic profiles for HNO reactions with met-myoglobin were obtained, as well as EPR evidence from carboxy-PTIO experiments. Importantly, the product HNO counteracted NO-induced as well as hypoxia-induced stabilization of the tumor-suppressor HIF-1α in cancer cells. The functional disruption of neovascularization by HNO produced by this metallonitrosyl complex was demonstrated in an in vitro angiogenesis model. This behavior is consistent with HNO biochemistry and contrasts with NO-mediated stabilization of HIF-1α. Together, these results demonstrate for the first time thiol-dependent production of HNO by a ruthenium complex and subsequent destabilization of HIF-1α. This work suggests that the complex warrants further investigation as a promising antiangiogenesis agent for the treatment of cancer.
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http://dx.doi.org/10.1021/acschembio.6b00222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949585PMC
July 2016

[Fe(CN)5(isoniazid)](3-): an iron isoniazid complex with redox behavior implicated in tuberculosis therapy.

J Inorg Biochem 2014 Nov 12;140:236-44. Epub 2014 Aug 12.

Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom. Electronic address:

Tuberculosis has re-emerged as a worldwide threat, which has motivated the development of new drugs. The antituberculosis complex Na3[Fe(CN)5(isoniazid)] (IQG607) in particular is of interest on account of its ability to overcome resistance. IQG607 has the potential for redox-mediated-activation, in which an acylpyridine (isonicotinoyl) radical could be generated without assistance from the mycobacterial KatG enzyme. Here, we have investigated the reactivity of IQG607 toward hydrogen peroxide and superoxide, well-known intracellular oxidizing agents that could play a key role in the redox-mediated-activation of this compound. HPLC, NMR and electronic spectroscopy studies showed a very fast oxidation rate for bound isoniazid, over 460-fold faster than free isoniazid oxidation. A series of EPR spin traps were used for detection of isonicotinoyl and derived radicals bound to iron. This is the first report for an isonicotinoyl radical bound to a metal complex, supported by (14)N and (1)H hyperfine splittings for the POBN and PBN trapped radicals. POBN and PBN exhibited average hyperfine coupling constants of aN=15.6, aH=2.8 and aN=15.4, aH=4.7, respectively, which are in close agreement to the isonicotinoyl radical. Radical generation is thought to play a major role in the mechanism of action of isoniazid and this work provides strong evidence for its production within IQG607, which, along with biological and chemical oxidation data, support a redox-mediated activation mechanism. More generally the concept of redox activation of metallo prodrugs could be applied more widely for the design of therapeutic agents with novel mechanisms of action.
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http://dx.doi.org/10.1016/j.jinorgbio.2014.08.002DOI Listing
November 2014

Activity of IQG-607, a new orally active compound, in a murine model of Mycobacterium tuberculosis infection.

Int J Antimicrob Agents 2012 Aug 27;40(2):182-5. Epub 2012 Jun 27.

Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF) and Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681 - Prédio 92A Tecnopuc, 90619-900 Porto Alegre, RS, Brazil.

We have previously demonstrated a potent in vitro inhibitory activity for two pentacyano(isoniazid)ferrate(II) compounds, namely IQG-607 and IQG-639, against the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase enzyme. In this study, the activity of these compounds was evaluated using an in vivo murine model of tuberculosis. Swiss mice were infected with M. tuberculosis H37Rv strain and then IQG-607 or IQG-639 (250 mg/kg) was administered for 28 days or 56 days. In addition, a dose-response study was performed with IQG-607 at 5, 10, 25, 50, 100, 200 and 250 mg/kg. The activity of test compounds was compared with that of the positive control drug isoniazid (INH) (25 mg/kg). After 28 days or 56 days of treatment, both IQG-607 and INH significantly reduced M. tuberculosis-induced splenomegaly as well as significantly diminishing the colony-forming units in the spleen and lungs. IQG-607 and INH ameliorated the lung macroscopic aspect, reducing lung lesions to a similar extent. However, IQG-639 did not significantly modify any evaluated parameter. Experiments using early and late controls of infection revealed a bactericidal activity for IQG-607. IQG-607 might well represent a good candidate for clinical development as a new antimycobacterial agent.
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http://dx.doi.org/10.1016/j.ijantimicag.2012.04.019DOI Listing
August 2012

Isoniazid metal complex reactivity and insights for a novel anti-tuberculosis drug design.

J Biol Inorg Chem 2012 Feb 28;17(2):275-83. Epub 2011 Sep 28.

Departamento de Química Orgânica e Inorgânica, Laboratório de Bioinorgânica, Universidade Federal do Ceará, P.O. Box 6021, Fortaleza, 60455-970, Brazil.

For over a decade, tuberculosis (TB) has been the leading cause of death among infectious diseases. Since the 1950s, isoniazid has been used as a front-line drug in the treatment of TB; however, resistant TB strains have limited its use. The major route of isoniazid resistance relies on KatG enzyme disruption, which does not promote an electron transfer reaction. Here, we investigated the reactivity of isoniazid metal complexes as prototypes for novel self-activating metallodrugs against TB with the aim to overcome resistance. Reactivity studies were conducted with hydrogen peroxide, hexacyanoferrate(III), and aquopentacyanoferrate(III). The latter species showed a preference for the inner-sphere electron transfer reaction pathway. Additionally, electron transfer reaction performed with either free isoniazid or (isoniazid)pentacyanoferrate(II) complex resulted in similar oxidized isoniazid derivatives as observed when the KatG enzyme was used. However, upon metal coordination, a significant enhancement in the formation of isonicotinic acid was observed compared with that of isonicotinamide. These results suggest that the pathway of a carbonyl-centered radical might be favored upon coordination to the Fe(II) owing to the π-back-bonding effect promoted by this metal center; therefore, the isoniazid metal complex could serve as a potential metallodrug. Enzymatic inhibition assays conducted with InhA showed that the cyanoferrate moiety is not the major player involved in this inhibition but the presence of isoniazid is required in this process. Other isoniazid metal complexes, [Ru(CN)(5)(izd)](3-) and [Ru(NH(3))(5)(izd)](2+) (where izd is isoniazid), were also unable to inhibit InhA, supporting our proposed self-activating mechanism of action. We propose that isoniazid reactivity can be rationally modulated by metal coordination chemistry, leading to the development of novel anti-TB metallodrugs.
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http://dx.doi.org/10.1007/s00775-011-0848-xDOI Listing
February 2012

Oxygen-sensing histidine-protein kinases: assays of ligand binding and turnover of response-regulator substrates.

Methods Enzymol 2008 ;437:173-89

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Heme-based sensors are a recently discovered functional class of heme proteins that serve to detect physiological fluctuations in oxygen (O(2)), carbon monoxide (CO), or nitric oxide (NO). Many of these modular sensors detect heme ligands by coupling a histidine-protein kinase to a heme-binding domain. They typically bind O2, CO, and NO but respond only to one of these ligands. Usually, they are active in the ferrous unliganded state but are switched off by saturation with O2. The heme-binding domains of these kinases are quite varied. They may feature a PAS fold, as in the Bradyrhizobium japonicum and Sinorhizobium melitoti FixL proteins, or a GAF fold, as in the Mycobacterium tuberculosis DevS and DosT proteins. Alternative folds, such as HNOB (also H-NOX), have also been noted for such signal-transducing kinases, although these classes are less well studied. Histidine-protein kinases function in partnership with cognate response-regulator substrate(s): usually transcription factors that they activate by phosphorylation. For example, FixL proteins specifically phosphorylate their FixJ partners, and DevS and DosT proteins phosphorylate DevR in response to hypoxia. We present methods for purifying these sensors and their protein substrates, verifying the quality of the preparations, determining the K(d) values for binding of ligand and preparing sensors of known saturation, and measuring the rates of turnover (k(cat)) of the protein substrate by sensors of known heme status.
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http://dx.doi.org/10.1016/S0076-6879(07)37010-9DOI Listing
July 2008

DosT and DevS are oxygen-switched kinases in Mycobacterium tuberculosis.

Protein Sci 2007 Aug 28;16(8):1708-19. Epub 2007 Jun 28.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.

Exposure of Mycobacterium tuberculosis to hypoxia is known to alter the expression of many genes, including ones thought to be involved in latency, via the transcription factor DevR (also called DosR). Two sensory kinases, DosT and DevS (also called DosS), control the activity of DevR. We show that, like DevS, DosT contains a heme cofactor within an N-terminal GAF domain. For full-length DosT and DevS, we determined the ligand-binding parameters and the rates of ATP reaction with the liganded and unliganded states. In both proteins, the heme state was coupled to the kinase such that the unliganded, CO-bound, and NO-bound forms were active, but the O(2)-bound form was inactive. Oxygen-bound DosT was unusually inert to oxidation to the ferric state (half life in air >60 h). Though the kinase activity of DosT was unaffected by NO, this ligand bound 5000 times more avidly than O(2) to DosT (K(d) [NO] approximately 5 nM versus K(d) [O(2)] = 26 microM). These results demonstrate direct and specific O(2) sensing by proteins in M. tuberculosis and identify for the first time a signal ligand for a sensory kinase from this organism. They also explain why exposure of M. tuberculosis to NO donors under aerobic conditions can give results identical to hypoxia, i.e., NO saturates DosT, preventing O(2) binding and yielding an active kinase.
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http://dx.doi.org/10.1110/ps.072897707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2203369PMC
August 2007

A memory of oxygen binding explains the dose response of the heme-based sensor FixL.

Biochemistry 2007 May 8;46(21):6249-57. Epub 2007 May 8.

Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038, USA.

Bradyrhizobium japonicum FixL is a modular oxygen sensor that directs adaptations to hypoxia by coupling the status of a heme-binding domain to a histidine-protein kinase activity. The oxygen-bound form is the "off-state". The unliganded form is the "on-state" active kinase that phosphorylates a transcription factor, FixJ. We have developed methods to optimize the kinase reactions of FixL and measure the turnover rates (kcat) for reactions catalyzed by highly inhibited states of this sensor at constant, precisely known oxygen saturations. The resulting oxygen dose-response curve shows that an in vitro system with FixL and the response regulator FixJ as its only proteins manifests such a sharp ligand response that, when the proportion of deoxy-FixL decreases less than 3-fold, the kinase activity drops over 50-fold, and by the time the deoxy-FixL declines just 8-fold, the activity is inhibited over 1100-fold. This response is entirely reversible and similar to that reported for the in vivo hypoxic induction of FixLJ-regulated genes. FixL binds oxygen noncooperatively. When complexed with FixJ, FixL is dimeric in both oxy and deoxy states. Therefore traditional models involving cooperative binding of ligand or robust allosteric regulation cannot account for the extremely nonlinear kinase response to the heme saturation. This response, however, can be explained by a form of enzyme hysteresis with the simple assumptions that (i) on association of oxygen with the heme, the kinase is rapidly switched off; (ii) after dissociation of oxygen, the kinase remains inhibited longer than the average time that it takes a deoxy-heme to encounter an oxygen molecule at most oxygen saturations.
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http://dx.doi.org/10.1021/bi7003334DOI Listing
May 2007

A proximal arginine R206 participates in switching of the Bradyrhizobium japonicum FixL oxygen sensor.

J Mol Biol 2006 Jun 11;360(1):80-9. Epub 2006 May 11.

Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9038, USA.

In oxygen-sensing PAS domains, a conserved polar residue on the proximal side of the heme cofactor, usually arginine or histidine, interacts alternately with the protein in the "on-state" or the heme edge in the "off-state" but does not contact the bound ligand directly. We assessed the contributions of this residue in Bradyrhizobium japonicum FixL by determining the effects of an R206A substitution on the heme-PAS structure, ligand affinity, and regulatory capacity. The crystal structures of the unliganded forms of the R206A and wild-type BjFixL heme-PAS domains were similar, except for a more ruffled porphyrin ring in R206A BjFixL and a relaxation of the H214 residue and heme propionate 7 due to their lost interactions. The oxygen affinity of R206A BjFixL (Kd approximately 350 microM) was 2.5 times lower than that of BjFixL, and this was due to a higher off-rate constant for the R206A variant. The enzymatic activities of the unliganded "on-state" forms, either deoxy or met-R206A BjFixL, were comparable to each other and slightly lower (twofold less) than those of the corresponding BjFixL species. The most striking difference between the two proteins was in the enzymatic activities of the liganded "off-state" forms. In particular, saturation with a regulatory ligand (the Fe(III) form with cyanide) caused a >2000-fold inhibition of the BjFixL phosphorylation of BjFixJ, but a 140-fold inhibition of this catalytic activity in R206A BjFixL. Thus, in oxygen-sensing PAS domains, the interactions of polar residues with the heme edge couple the heme-binding domain to a transmitter during signal transduction.
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http://dx.doi.org/10.1016/j.jmb.2006.04.054DOI Listing
June 2006

Oxygen blocks the reaction of the FixL-FixJ complex with ATP but does not influence binding of FixJ or ATP to FixL.

Biochemistry 2005 Nov;44(46):15359-65

Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038, USA.

The RmFixL-RmFixJ oxygen signal transduction system ensures that a cascade of the Sinorhizobium meliloti nitrogen fixation genes is induced as the concentration of O2 drops below 50 microM in symbiotic nodules. Deoxy-RmFixL is a histidine protein kinase that catalyzes a phosphoryl transfer from ATP to the aspartate 54 residue of RmFixJ; RmFixJ is a response regulator that becomes activated as a transcription factor by phosphorylation. Association of O2 with a heme-binding domain in RmFixL triggers a conformational change that inhibits its kinase activity. Here we consider whether this inhibition is achieved by disrupting binding of either of the substrates, i.e., RmFixJ or ATP, to the RmFixL kinase. The ATP affinities of the oxy and deoxy states were compared via competition of ATP against TNP-nucleotide fluorophores. The influence of O2 on formation of the RmFixL-RmFixJ complex was investigated by fluorescence polarization. Oxygen dramatically inhibited the reaction of the RmFixL-RmFixJ complex with ATP but affected neither ATP binding (Kd approximately 100 microM) nor RmFixL-RmFixJ complex formation (Kd approximately 4 microM), indicating that inhibition of the kinase by the oxy-heme in RmFixL is achieved by inactivating the catalytic site, rather than by blocking the association of this enzyme with either of its substrates. An 8-fold enhancement of the rate of reaction of RmFixL with ATP in a deoxy-RmFixL-D54N RmFixJ complex, compared to that in isolated deoxy-RmFixL, exposes the strength of the allosteric effect of RmFixJ on the reaction. These results clarify the mechanistic roles of the signal and regulatory partner in this signal transduction system.
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http://dx.doi.org/10.1021/bi051661hDOI Listing
November 2005
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