Antimicrobial and anticancer properties of carbon monoxide releasing molecules of the fac-[Re(CO)3(N-N)L]+ family.

The toxicity profile of fac-[Re(CO)3(N-N)L]+ complexes against microbial and tumoral cells has been extensively studied, primarily focusing on modifications to the bidentate diimine (N-N) ligand. However, less attention has been paid to modifications of the axial ligand L, which is perpendicular to the Re-N-N plane. This study reveals that the high toxicity of the fac-[Re(CO)3(bpy)(Ctz)]+ complex may be attributed to the structural effect of the trityl (CPh3) group present in clotrimazole, as removal of phenyl rings causes a significant decrease in the activity against Staphylococcus aureus (S. aureus). Moreover, substitution of the 1-tritylimidazole ligand by the structurally related ligands PPh3 and PCy3 maintains similarly high activity levels. These findings contribute to understanding the interactions of toxic complexes with bacterial membranes, suggesting that the ligand structures play a crucial role in inhibiting cell wall synthesis processes, potentially including Lipid II synthesis. Compounds with Ph3E (E = C-imidazole; P) groups also showed to be 10 times more toxic than cisplatin against three mammalian cell lines (IC50: 2-4 µM). In contrast, the analogue 1-benzylimidazole and 1-tert-butylimidazole derivatives were as toxic as cisplatin. We observed that the decomposition of the [Re(I)(CO)3] fragment inside mammalian cell lines liberates CO, which is expected to exert biological effects. Therefore, compounds of this family possessing the structural motif Ph3E seem to combine high antimicrobial and antitumoral activities, the latter being much higher than that of cisplatin.

Carbon Monoxide Modulation of Microglia-Neuron Communication: Anti-Neuroinflammatory and Neurotrophic Role.

Microglia, the 'resident immunocompetent cells' of the central nervous system (CNS), are key players in innate immunity, synaptic refinement and homeostasis. Dysfunctional microglia contribute heavily to creating a toxic inflammatory milieu, a driving factor in the pathophysiology of several CNS disorders. Therefore, strategies to modulate the microglial function are required to tackle exacerbated tissue inflammation. Carbon monoxide (CO), an endogenous gaseous molecule produced by the degradation of haem, has anti-inflammatory, anti-apoptotic, and pro-homeostatic and cytoprotective roles, among others. ALF-826A, a novel molybdenum-based CO-releasing molecule, was used for the assessment of neuron-microglia remote communication. Primary cultures of rat microglia and neurons, or the BV-2 microglial and CAD neuronal murine cell lines, were used to study the microglia-neuron interaction. An approach based on microglial-derived conditioned media in neuronal culture was applied. Medium derived from CO-treated microglia provided indirect neuroprotection against inflammation by limiting the lipopolysaccharide (LPS)-induced expression of reactivity markers (CD11b), the production of reactive oxygen species (ROS) and the secretion of inflammatory factors (TNF-α, nitrites). This consequently prevented neuronal cell death and maintained neuronal morphology. In contrast, in the absence of inflammatory stimulus, conditioned media from CO-treated microglia improved neuronal morphological complexity, which is an indirect manner of assessing neuronal function. Likewise, the microglial medium also prevented neuronal cell death induced by pro-oxidant tert-Butyl hydroperoxide (t-BHP). ALF-826 treatment reinforced microglia secretion of Interleukin-10 (IL-10) and adenosine, mediators that may protect against t-BHP stress in this remote communication model. Chemical inhibition of the adenosine receptors A2A and A1 reverted the CO-derived neuroprotective effect, further highlighting a role for CO in regulating neuron-microglia communication via purinergic signalling. Our findings indicate that CO has a modulatory role on microglia-to-neuron communication, promoting neuroprotection in a non-cell autonomous manner. CO enhances the microglial release of neurotrophic factors and blocks exacerbated microglial inflammation. CO improvement of microglial neurotrophism under non-inflammatory conditions is here described for the first time.

Carbon Monoxide-Neuroglobin Axis Targeting Metabolism Against Inflammation in BV-2 Microglial Cells.

Microglia are the immune competent cell of the central nervous system (CNS), promoting brain homeostasis and regulating inflammatory response against infection and injury. Chronic or exacerbated neuroinflammation is a cause of damage in several brain pathologies. Endogenous carbon monoxide (CO), produced from the degradation of heme, is described as anti-apoptotic and anti-inflammatory in several contexts, including in the CNS. Neuroglobin (Ngb) is a haemoglobin-homologous protein, which upregulation triggers antioxidant defence and prevents neuronal apoptosis. Thus, we hypothesised a crosstalk between CO and Ngb, in particular, that the anti-neuroinflammatory role of CO in microglia depends on Ngb. A novel CO-releasing molecule (ALF826) based on molybdenum was used for delivering CO in microglial culture.BV-2 mouse microglial cell line was challenged with lipopolysaccharide (LPS) for triggering inflammation, and after 6 h ALF826 was added. CO exposure limited inflammation by decreasing inducible nitric oxide synthase (iNOS) expression and the production of nitric oxide (NO) and tumour necrosis factor-α (TNF-α), and by increasing interleukine-10 (IL-10) release. CO-induced Ngb upregulation correlated in time with CO's anti-inflammatory effect. Moreover, knocking down Ngb reversed the anti-inflammatory effect of CO, suggesting that dependents on Ngb expression. CO-induced Ngb upregulation was independent on ROS signalling, but partially dependent on the transcriptional factor SP1. Finally, microglial cell metabolism is also involved in the inflammatory response. In fact, LPS treatment decreased oxygen consumption in microglia, indicating a switch to glycolysis, which is associated with a proinflammatory. While CO treatment increased oxygen consumption, reverting LPS effect and indicating a metabolic shift into a more oxidative metabolism. Moreover, in the absence of Ngb, this phenotype was no longer observed, indicating Ngb is needed for CO's modulation of microglial metabolism. Finally, the metabolic shift induced by CO did not depend on alteration of mitochondrial population. In conclusion, neuroglobin emerges for the first time as a key player for CO signalling against exacerbated inflammation in microglia.

Study of the interactions of bovine serum albumin with a molybdenum(II) carbonyl complex by spectroscopic and molecular simulation methods.

Therapy with inhaled carbon monoxide (CO) is being tested in human clinical trials, yet the alternative use of prodrugs, CO-Releasing Molecules (CORMs), is conceptually advantageous. These molecules are designed to release carbon monoxide in specific tissues, in response to some locally expressed stimulus, where CO can trigger a cytoprotective response. The design of such prodrugs, mostly metal carbonyl complexes, must consider their ADMET profiles, including their interaction with transport plasma proteins. However, the molecular details of this interaction remain elusive. To shed light into this matter, we focused on the CORM prototype [Mo(η5-Cp)(CH2COOH)(CO)3] (ALF414) and performed a detailed molecular characterization of its interaction with bovine serum albumin (BSA), using spectroscopic and computational methods. The experimental results show that ALF414 partially quenches the intrinsic fluorescence of BSA without changing its secondary structure. The interaction between BSA and ALF414 follows a dynamic quenching mechanism, indicating that no stable complex is formed between the protein Trp residues and ALF414. The molecular dynamics simulations are in good agreement with the experimental results and confirm the dynamic and unspecific character of the interaction between ALF414 and BSA. The simulations also provide important insights into the nature of the interactions of this CORM prototype with BSA, which are dominated by hydrophobic contacts, with a contribution from hydrogen bonding. This kind of information is useful for future CORM design.

Aluminum Doped MCM-41 Nanoparticles as Platforms for the Dual Encapsulation of a CO-Releasing Molecule and Cisplatin.

Mesoporous silica Al-MCM-41 nanoparticles have been used, for the first time, as vehicles for the single and dual encapsulation of the cationic CO-releasing molecule (CORM) [Mn(1,4,7-triazacyclononane)(CO)3]+ (ALF472+) and the well-known antineoplastic drug, cis-[PtCl2(NH3)2] (cisplatin). Thus, two new hybrid materials, namely, ALF472@Al-MCM-41 and ALF472-cisplatin@Al-MCM-41, have been isolated and fully characterized. The results reveal that the presence of CORM molecules enhances cisplatin loading 3-fold, yielding a cargo of 0.45 mmol g-1 of ALF472+ and 0.12 mmol g-1 of the platinum complex for ALF472-cisplatin@Al-MCM-41. It is worth noting that ALF472@Al-MCM-41 shows a good dispersion in phosphate buffered saline solution, while the dual hybrid material slightly aggregates in this simulated physiological medium (hydrodynamic size: 112 ± 23 and 336 ± 50 nm, respectively). In addition, both hybrid materials (ALF472@Al-MCM-41 and ALF472-cisplatin@Al-MCM-41) behave as photoactive CO-releasing materials, delivering 0.25 and 0.11 equiv of CO, respectively, after 24 h and exhibiting a more controlled CO delivery than that of the free CORM. Finally, metal leaching studies have confirmed the good retention capacity of Al-MCM-41 toward the potentially toxic manganese fragments (86% of retention after 72 h) as well as the low release of cisplatin (ca. 7% after 72 h).

Examining the antimicrobial activity and toxicity to animal cells of different types of CO-releasing molecules.

Transition metal carbonyl complexes used as CO-releasing molecules (CORMs) for biological and therapeutic applications may exhibit interesting antimicrobial activity. However, understanding the chemical traits and mechanisms of action that rule this activity is required to establish a rationale for the development of CORMs into useful antibiotics. In this work the bactericidal activity, the toxicity to eukaryotic cells, and the ability of CORMs to deliver CO to bacterial and eukaryotic cells were analysed for a set of seven CORMs that differ in the transition metal, ancillary ligands and the CO release profile. Most of these CORMs exhibited bactericidal properties that decrease in the following order: CORM-2 > CORM-3 > ALF062 > ALF850 > ALF186 > ALF153 > [Fe(SBPy3)(CO)](BF4)2. A similar yet not entirely coincident decreasing order was found for their induction of intracellular reactive oxygen species (ROS) in E. coli. In contrast, studies in model animal cells showed that for any given CORM, the level of intracellular ROS generated was negligible when compared with that measured inside bacteria. Importantly, these CORMs were in general not toxic to eukaryotic cells, namely murine macrophages, kidney LLC-PK1 epithelial cells, and liver cell line HepG2. CORM-2 and CORM-3 delivered CO to the intracellular space of both E. coli and the two types of tested eukaryotic cells, yet toxicity was only elicited in the case of E. coli. CO delivered by ALF186 into the intercellular space did not enter E. coli cells and the compound was not toxic to either bacteria or to eukaryotic cells. The Fe(ii) carbonyl complex [Fe(SBPy3)(CO)](2+) had the reverse, undesirable toxicity profile, being unexpectedly toxic to eukaryotic cells and non-toxic to E. coli. ALF153, the most stable complex in the whole set, was essentially devoid of toxicity or ROS induction ability in all cells. These results suggest that CORMs have a relevant therapeutic potential as antimicrobial drugs since (i) they can show opposite toxicity profiles towards bacteria and eukaryotic cells; (ii) their activity can be modulated through manipulation of the ancillary ligands, as shown with the three {Ru(CO)3}(2+) and two zerovalent Mo based CORMs; and (iii) their toxicity to eukaryotic cells can be made acceptably low. With this new approach, this work contributes to the understanding of the roots of the bactericidal action of CORMs and helps in establishing strategies for their development into a new class of antibiotics.

An N-Acetyl Cysteine Ruthenium Tricarbonyl Conjugate Enables Simultaneous Release of CO and Ablation of Reactive Oxygen Species.

We have designed and synthesised a [Ru(CO)3 Cl2 (NAC)] pro-drug that features an N-acetyl cysteine (NAC) ligand. This NAC carbon monoxide releasing molecule (CORM) conjugate is able to simultaneously release biologically active CO and to ablate the concurrent formation of reactive oxygen species (ROS). Complexes of the general formulae [Ru(CO)3 (L)3 ](2+) , including [Ru(CO)3 Cl(glycinate)] (CORM-3), have been shown to produce ROS through a water-gas shift reaction, which contributes significantly, for example, to their antibacterial activity. In contrast, NAC-CORM conjugates do not produce ROS or possess antibacterial activity. In addition, we demonstrate the synergistic effect of CO and NAC both for the inhibition of nitric oxide (formation) and in the expression of tumour-necrosis factor (TNF)-α. This work highlights the advantages of combining a CO-releasing scaffold with the anti-oxidant and anti-inflammatory drug NAC in a unique pro-drug.

Spontaneous CO release from Ru(II)(CO)2-protein complexes in aqueous solution, cells, and mice.

We demonstrate that Ru(II)(CO)2-protein complexes, formed by the reaction of the hydrolytic decomposition products of [fac-RuCl(κ(2)-H2NCH2CO2)(CO)3] (CORM-3) with histidine residues exposed on the surface of proteins, spontaneously release CO in aqueous solution, cells, and mice. CO release was detected by mass spectrometry (MS) and confocal microscopy using a CO-responsive turn-on fluorescent probe. These findings support our hypothesis that plasma proteins act as CO carriers after in vivo administration of CORM-3. CO released from a synthetic bovine serum albumin (BSA)-Ru(II)(CO)2 complex leads to downregulation of the cytokines interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α in cancer cells. Finally, administration of BSA-Ru(II)(CO)2 in mice bearing a colon carcinoma tumor results in enhanced CO accumulation at the tumor. Our data suggest the use of Ru(II)(CO)2-protein complexes as viable alternatives for the safe and spatially controlled delivery of therapeutic CO in vivo.

Carbon monoxide abrogates ischemic insult to neuronal cells via the soluble guanylate cyclase-cGMP pathway.

PURPOSE: Carbon monoxide (CO) is an accepted cytoprotective molecule. The extent and mechanisms of protection in neuronal systems have not been well studied. We hypothesized that delivery of CO via a novel releasing molecule (CORM) would impart neuroprotection in vivo against ischemia-reperfusion injury (IRI)-induced apoptosis of retinal ganglion cells (RGC) and in vitro of neuronal SH-SY5Y-cells via activation of soluble guanylate-cyclase (sGC). METHODS: To mimic ischemic respiratory arrest, SH-SY5Y-cells were incubated with rotenone (100 nmol/L, 4 h) ± CORM ALF186 (10-100 µmol/L) or inactivated ALF186 lacking the potential of releasing CO. Apoptosis and reactive oxygen species (ROS) production were analyzed using flow-cytometry (Annexin V, mitochondrial membrane potential, CM-H2DCFDA) and Western blot (Caspase-3). The impact of ALF186± respiratory arrest on cell signaling was assessed by measuring expression of nitric oxide synthase (NOS) and soluble guanylate-cyclase (sGC) and by analyzing cellular cGMP levels. The effect of ALF186 (10 mg/kg iv) on retinal IRI in Sprague-Dawley rats was assessed by measuring densities of fluorogold-labeled RGC after IRI and by analysis of apoptosis-related genes in retinal tissue. RESULTS: ALF186 but not inactivated ALF186 inhibited rotenone-induced apoptosis (Annexin V positive cells: 25 ± 2% rotenone vs. 14 ± 1% ALF186+rotenone, p<0.001; relative mitochondrial membrane potential: 17 ± 4% rotenone vs. 55 ± 3% ALF186+rotenone, p<0.05). ALF186 increased cellular cGMP levels (33±5 nmol/L vs. 23±3 nmol/L; p<0.05) and sGC expression. sGC-inhibition attenuated ALF186-mediated protection (relative mitochondrial membrane potential: 55±3% ALF186+rotenone vs. 20 ± 1% ODQ + ALF186+rotenone, p<0.05). ALF186 protected RGC in vivo (IRI 1255 ± 327 RGC/mm(2) vs. ALF186 + IRI 2036 ± 83; p<0.05) while sGC inhibition abolished the protective effects of ALF186 (ALF186 + IRI 2036 ± 83 RGC/mm(2) vs. NS-2028 + ALF186 + IRI 1263 ± 170, p<0.05). CONCLUSIONS: The CORM ALF186 inhibits IRI-induced neuronal cell death via activation of sGC and may be a useful treatment option for acute ischemic insults to the retina and the brain.

Characterization of a versatile organometallic pro-drug (CORM) for experimental CO based therapeutics.

The complex fac-[Mo(CO)(3)(histidinate)]Na has been reported to be an effective CO-Releasing Molecule in vivo, eliciting therapeutic effects in several animal models of disease. The CO releasing profile of this complex in different settings both in vitro and in vivo reveals that the compound can readily liberate all of its three CO equivalents under biological conditions. The compound has low toxicity and cytotoxicity and is not hemolytic. CO release is accompanied by a decrease in arterial blood pressure following administration in vivo. We studied its behavior in solution and upon the interaction with proteins. Reactive oxygen species (ROS) generation upon exposure to air and polyoxomolybdate formation in soaks with lysozyme crystals were observed as processes ensuing from the decomposition of the complex and the release of CO.

Heterometallic complexes involving iron(II) and rhenium(VII) centers connected by mu-oxido bridges.

The reaction of FeI(2) with two equivalents of AgReO(4) in acetonitrile leads to yellow, crystalline Fe(ReO(4))(2)(CH(3)CN)(3) (1), and the treatment of 1 with four equivalents of CpFe(CO)(2)CN gives orange, crystalline Fe(ReO(4))(2)[CpFe(CO)(2)(CN)](4) (2). Compound 2 can also be prepared in one step by the reaction of FeI(2), AgReO(4) and CpFe(CO)(2)CN in dichloromethane. The structure of 1 consists of infinite chains in which alternating {Fe(CH(3)CN)(4)} and {Fe(ReO(4))(2)(CH(3)CN)(2)} units are linked by perrhenate anions to form a (-ReO(2)-O-Fe(ReO(4))(2)(CH(3)CN)(2)-O-ReO(2)-O-Fe(CH(3)CN)(4)-O-)(n) molecular wire. The structure of 2 shows a monomeric iron complex with a slightly distorted octahedral coordination environment consisting of four organometallic complexes coordinated in the equatorial plane via the cyanide groups and two monodentate perrhenates in the corresponding apical positions. Both compounds were further characterised in the solid state by IR spectroscopy, thermogravimetric analysis and magnetic susceptibility measurements. The magnetic data indicate that 1 behaves as a ferrimagnetic chain with 3D ordering below 8 K due to inter-chain interactions. Compound 2 has antiferromagnetic interactions within the Fe(ReO(4))(2)[CpFe(CO)(2)(CN)](4) clusters.