The Enigma of Norbormide, a Rattus-Selective Toxicant.

Norbormide (NRB) is a Rattus-selective toxicant, which was serendipitously discovered in 1964 and formerly marketed as an eco-friendly rodenticide that was deemed harmless to non-Rattus species. However, due to inconsistent efficacy and the emergence of second-generation anticoagulants, its usage declined, with registration lapsing in 2003. NRBs' lethal action in rats entails irreversible vasoconstriction of peripheral arteries, likely inducing cardiac damage: however, the precise chain of events leading to fatality and the target organs involved remain elusive. This unique contractile effect is exclusive to rat arteries and is induced solely by the endo isomers of NRB, hinting at a specific receptor involvement. Understanding NRB's mechanism of action is crucial for developing species-selective toxicants as alternatives to the broad-spectrum ones currently in use. Recent research efforts have focused on elucidating its cellular mechanisms and sites of action using novel NRB derivatives. The key findings are as follows: NRB selectively opens the rat mitochondrial permeability transition pore, which may be a factor that contributes to its lethal effect; it inhibits rat vascular KATP channels, which potentially controls its Rattus-selective vasoconstricting activity; and it possesses intracellular binding sites in both sensitive and insensitive cells, as revealed by fluorescent derivatives. These studies have led to the development of a prodrug with enhanced pharmacokinetic and toxicological profiles, which is currently undergoing registration as a novel efficacious eco-sustainable Rattus-selective toxicant. The NRB-fluorescent derivatives also show promise as non-toxic probes for intracellular organelle labelling. This review documents in more detail these developments and their implications.

Nature-Inspired Peptide Antifouling Biocide: Coating Compatibility, Field Validation, and Environmental Stability.

This study reports the development of a class of eco-friendly antifouling biocides based on a cyclic dipeptide scaffold, 2,5-diketopiperazine (2,5-DKP). The lead compound cyclo(N-Bip-l-Arg-N-Bip-l-Arg) (1) was synthesized in gram amounts and used to assess the compatibility with an ablation/hydration coating, efficacy against biofouling, and biodegradation. Leaching of 1 from the coating into seawater was assessed via a rotating drum method, revealing relatively stable and predictable leaching rates under dynamic shear stress conditions (36.1 ± 19.7 to 25.2 ± 9.1 ng-1 cm-2 day-1) but low or no leaching under static conditions. The coatings were further analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS), with 1 seen to localize at the surface of the coating in a surfactant-like fashion. When coatings were deployed in the ocean, detectable reductions in biofouling development were measured for up to 11 weeks. After this time, biofouling overwhelmed the performance of the coating, consistent with leaching kinetics. Biodegradation of 1 in seawater was assessed using theoretical oxygen demand and analytical quantification. Masking effects were observed at higher concentrations of 1 due to antimicrobial properties, but half-lives were calculated ranging from 13.4 to 16.2 days. The results can rationally inform future development toward commercial antifouling products.

Effect of regio- and stereoisomerism on antifouling 2,5-diketopiperazines.

Marine biofouling is a problem that plagues all maritime industries at vast economic and environmental cost. Previous and current methods to prevent biofouling have employed the use of heavy metals and other toxic or highly persistent chemicals, and these methods are now coming under immense regulatory pressure. Recent studies have illustrated the potential of nature-inspired tetrasubstituted 2,5-diketopiperazines (2,5-DKPs) as eco-friendly marine biocides for biofouling control. These highly active symmetrically substituted 2,5-DKPs can be generated by combining structural motifs from cationic innate defence peptides and natural marine antifoulants. A balance between a threshold hydrophobic contribution and sufficient cationic charge has been established as key for bioactivity, and our current study further increases understanding of the antifouling mechanism by investigating the effect of both regio- and stereochemistry. Novel synthetic routes for the generation of unsymmetrical 2,5-DKPs were developed and a library of nine compounds was prepared. The compounds were screened against a series of four model macrofouling organisms (Ciona savignyi, Mytilus galloprovincialis, Spirobranchus cariniferus, and Undaria pinnatifida). Several of the evaluated compounds displayed inhibitory activity at sub-micromolar concentrations. The structural contributions to antifouling bioactivity were studied using NMR spectroscopy and molecular modelling, revealing a strong dependence on a stable amphiphilic solution structure regardless of substitution pattern.

Stereochemical Effects on the Antimicrobial Properties of Tetrasubstituted 2,5-Diketopiperazines.

Antimicrobial drug resistance is a looming health crisis facing us in the modern era, and new drugs are urgently needed to combat this growing problem. Synthetic mimics of antimicrobial peptides have recently emerged as a promising class of compounds for the treatment of persistent microbial infections. In the current study, we investigate five cyclic N-alkylated amphiphilic 2,5-diketopiperazines against 15 different strains of bacteria and fungi, including drug-resistant clinical isolates. Several of the 2,5-diketopiperazines displayed activities similar or superior to antibiotics currently in clinical use, with activities coupled to both the cationic and hydrophobic substituents. All possible stereoisomers of the lead peptide were prepared, and the effects of stereochemistry and amphiphilicity were investigated via 1D and 2D NMR spectroscopy, solution dynamics, and membrane interaction modeling. Clear differences in solution structures and membrane interaction potentials explain the differences seen in the bioactivity and physicochemical properties of each stereoisomer.

Nitrobenzoxadiazole derivatives of the rat selective toxicant norbormide as fluorescent probes for live cell imaging.

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats, but relatively harmless to other rodents and mammals. As a vasoactive agent, NRB induces a species-specific vasocontractile effect that is restricted to the peripheral arteries of the rat. Despite the precise mechanisms behind this phenomenon having yet to be fully clarified, it is postulated that the molecular target of NRB could be located within the plasma membrane of rat peripheral artery myocytes (e.g. rat caudal artery myocytes). As such, the primary objective of this study was to develop a fluorescently labelled derivative of NRB to investigate its subcellular distribution/localization in both NRB-sensitive (freshly isolated rat caudal artery myocytes, FIRCAMs) and NRB-insensitive (human hepatic stellate, LX2) cells. Of the examples prepared, lead structure endo-NRB-NBD-bPA subsequently demonstrated retention of the parent toxicant's pharmacological profile (in terms of its ability to induce both a vasocontractile response in rat caudal artery rings in vitro, and a lethal end-point in rats in vivo). Endo-NRB-NBD-bPA was also shown to be significantly less permeable (an integral feature in the design of fluorescent probes targeting cell-surface receptors) to both LX2 cells and FIRCAMs. Disappointingly, no fluorescence could be observed on the plasma membrane of FIRCAMs stained with endo-NRB-NBD-bPA.

Towards eco-friendly marine antifouling biocides - Nature inspired tetrasubstituted 2,5-diketopiperazines.

Marine biofouling plagues all maritime industries at vast economic and environmental cost. Previous and most current methods to control biofouling have employed highly persistent toxins and heavy metals, including tin, copper, and zinc. These toxic methods are resulting in unacceptable environmental harm and are coming under immense regulatory pressure. Eco-friendly alternatives are urgently required to effectively mitigate the negative consequence of biofouling without causing collateral harm. Amphiphilic micropeptides have recently been shown to exhibit excellent broad-spectrum antifouling activity, with a non-toxic mode of action and innate biodegradability. The present work focused on incorporating the pharmacophore derived from amphiphilic micropeptides into a 2,5-diketopiperazine (DKP) scaffold. This privileged structure is present in a vast number of natural products, including marine natural product antifoulants, and provides advantages of synthetic accessibility and adaptability. A novel route to symmetrical tetrasubstituted DKPs was developed and a library of amphiphilic 2,5-DKPs were subsequently synthesised. These biodegradable compounds were demonstrated to be potent marine antifoulants displaying broad-spectrum activity in the low micromolar range against a range of common marine fouling organisms. The outcome of planned coating and field trials will dictate the future development of the lead compounds.

Disruption of Metallostasis in the Anaerobic Human Pathogen Fusobacterium nucleatum by the Zinc Ionophore PBT2.

The Gram-negative anaerobe Fusobacterium nucleatum is an opportunistic human pathogen, most frequently associated with periodontal disease through dental biofilm formation and, increasingly, with colorectal cancer development and progression. F. nucleatum infections are routinely treated by broad-spectrum ß-lactam antibiotics and metronidazole. However, these antibiotics can negatively impact the normal microflora. Therefore, the development of novel narrow-spectrum antimicrobials active against anaerobic pathogens is of great interest. Here, we examined the antimicrobial Zn ionophore PBT2, an 8-hydroxyquinoline analogue with metal chelating properties, against a single type isolate F. nucleatum ATCC 25586. PBT2-Zn was a potent inhibitor of growth and exhibited synergistic bactericidal (>3-log10 killing) activity at 5× MIC in planktonic cells, and at the MIC in biofilms grown in vitro. Physiological and transcriptional analyses uncovered a strong cellular response relating to Zn and Fe homeostasis in PBT2-Zn treated cells across subinhibitory and inhibitory concentrations. At 1× MIC, PBT2 alone induced a 3.75-fold increase in intracellular Zn, whereas PBT2-Zn challenge induced a 19-fold accumulation of intracellular Zn after 2 h. A corresponding 2.1-fold loss of Fe was observed at 1× MIC. Transcriptional analyses after subinhibitory PBT2-Zn challenge (0.125 µg/mL and 200 µM ZnSO4) revealed significant differential expression of 15 genes at 0.5 h, and 12 genes at 1 h. Upregulated genes included those with roles in Zn homeostasis (e.g., a Zn-transporting ATPase and the Zn-sensing transcriptional regulator, smtB) and hemin transport (hmuTUV) to re-establish Fe homeostasis. A concentration-dependent protective effect was observed for cells pretreated with hemin (50 µg/mL) prior to PBT2-Zn challenge. The data presented here supports our proposal that targeting the disruption of metallostasis by Zn-translocating ionophores is a strategy worth investigating further for the treatment of Gram-negative anaerobic pathogens.

Norbormide-Based Probes and Their Application for Mitochondrial Imaging in Drosophila Melanogaster.

Fluorescent live imaging on Drosophila melanogaster is a microscopy technique in rapid expansion. The growing number of probes available to detect cellular components and the relatively easy genetic manipulation of fruit fly make this model one of the most used for in vivo analysis of several physiological and/or pathological processes. Here we describe the chemical synthesis of two norbormide-derived BODIPY-conjugated fluorescent probes (NRBMC009 and NRBZLW0047). Moreover, we describe the larval dissection method, and subsequent live imaging acquisition. Both probes are able to label mitochondria in different Drosophila larval tissues, which allows for the characterization of mitochondrial morphological alterations by using a simple and quick method that avoids the fixation artefacts that often occur in immunofluorescence studies.

C-2 derivatized 8-sulfonamidoquinolines as antibacterial compounds.

A series of C-2 derivatized 8-sulfonamidoquinolines were evaluated for their antibacterial activity against the common mastitis causative pathogens Streptococcus uberis, Staphylococcus aureus and Escherichia coli, both in the presence and absence of supplementary zinc (50 µM ZnSO4). The vast majority of compounds tested were demonstrated to be significantly more active against S. uberis when in the presence of supplementary zinc (MICs as low as 0.125 µg/mL were observed in the presence of 50 µM ZnSO4). Compounds 5, 34-36, 39, 58, 79, 82, 94 and 95 were shown to display the greatest antibacterial activity against S. aureus (MIC ≤ 8 µg/mL; both in the presence and absence of supplementary zinc), while compounds 56, 58 and 66 were demonstrated to also exhibit activity against E. coli (MIC ≤ 16 µg/mL; under all conditions). Compounds 56, 58 and 66 were subsequently confirmed to be bactericidal against all three mastitis pathogens studied, with MBCs (≥3log10 CFU/mL reduction) of ≤ 32 µg/mL (in both the presence and absence of 50 µM ZnSO4). To validate the sanitizing activity of compounds 56, 58 and 66, a quantitative suspension disinfection (sanitizer) test was performed. Sanitizing activity (>5log10 CFU/mL reduction in 5 min) was observed against both S. uberis and E. coli at compound concentrations as low as 1 mg/mL (compounds 56, 58 and 66), and against S. aureus at 1 mg/mL (compound 58); thereby validating the potential of compounds 56, 58 and 66 to function as topical sanitizers designed explicitly for use in non-human applications.

Distinct Patterns of Internalization of Different Calcitonin Gene-Related Peptide Receptors.

Calcitonin gene-related peptide (CGRP) is a neuropeptide that is involved in the transmission of pain. Drugs targeting CGRP or a CGRP receptor are efficacious in the treatment of migraine. The canonical CGRP receptor is a complex of a G protein-coupled receptor, the calcitonin-like receptor (CLR), with an accessory protein, receptor activity-modifying protein 1 (RAMP1). A second receptor, the AMY1 receptor, a complex of the calcitonin receptor with RAMP1, is a dual high-affinity receptor for CGRP and amylin. Receptor regulatory processes, such as internalization, are crucial for controlling peptide and drug responsiveness. Given the importance of CGRP receptor activity in migraine we compared the internalization profiles of both receptors for CGRP using novel fluorescent probes and a combination of live cell imaging, fixed cell imaging, and ELISA. This revealed stark differences in the regulation of each receptor with the AMY1 receptor unexpectedly showing little internalization.

Substituted sulfonamide bioisosteres of 8-hydroxyquinoline as zinc-dependent antibacterial compounds.

A series of substituted sulfonamide bioisosteres of 8-hydroxyquinoline were evaluated for their antibacterial activity against the common mastitis causative pathogens Streptococcus uberis, Staphylococcus aureus and Escherichia coli, both in the presence and absence of supplementary zinc. Compounds 9a-e, 10a-c, 11a-e, 12 and 13 were demonstrated to have MICs of 0.0625 µg/mL against S. uberis in the presence of 50 µM ZnSO4. Against S. aureus compounds 9g (MIC 4 µg/mL) and 11d (MIC 8 µg/mL) showed the greatest activity, whereas all compounds were found to be inactive against E. coli (MIC > 256 µg/mL); again in the presence of 50 µM ZnSO4. All compounds were demonstrated to be significantly less active in the absence of supplementary zinc. Compound 9g was subsequently confirmed to be bactericidal, with an MBC (≥3log10 cfu/mL reduction) of 0.125 µg/mL against S. uberis in the presence of 50 µM ZnSO4. To validate the sanitising activity of compound 9g in the presence of supplementary zinc, a quantitative suspension disinfection (sanitizer) test was performed. In this preliminary test, sanitizing activity (>5log10 reduction of CFU/mL in 5 min) was observed against S. uberis for compound 9g at concentrations as low as 1 mg/mL, validating the potential of this compound to function as a topical sanitizer against the major environmental mastitis-causing microorganism S. uberis.

Multiple Bactericidal Mechanisms of the Zinc Ionophore PBT2.

Globally, more antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance (AMR). The development of novel ionophores, a class of antimicrobials used exclusively in animals, holds promise as a strategy to replace or reduce essential human antimicrobials in veterinary practice. PBT2 is a zinc ionophore with recently demonstrated antibacterial activity against several Gram-positive pathogens, although the underlying mechanism of action is unknown. Here, we investigated the bactericidal mechanism of PBT2 in the bovine mastitis-causing pathogen, Streptococcus uberis In this work, we show that PBT2 functions as a Zn2+/H+ ionophore, exchanging extracellular zinc for intracellular protons in an electroneutral process that leads to cellular zinc accumulation. Zinc accumulation occurs concomitantly with manganese depletion and the production of reactive oxygen species (ROS). PBT2 inhibits the activity of the manganese-dependent superoxide dismutase, SodA, thereby impairing oxidative stress protection. We propose that PBT2-mediated intracellular zinc toxicity in S. uberis leads to lethality through multiple bactericidal mechanisms: the production of toxic ROS and the impairment of manganese-dependent antioxidant functions. Collectively, these data show that PBT2 represents a new class of antibacterial ionophores capable of targeting bacterial metal ion homeostasis and cellular redox balance. We propose that this novel and multitarget mechanism of PBT2 makes the development of cross-resistance to medically important antimicrobials unlikely.IMPORTANCE More antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance. Therefore, the elimination of antimicrobial crossover between human and veterinary medicine is of great interest. Unfortunately, the development of new antimicrobials is an expensive high-risk process fraught with difficulties. The repurposing of chemical agents provides a solution to this problem, and while many have not been originally developed as antimicrobials, they have been proven safe in clinical trials. PBT2, a zinc ionophore, is an experimental therapeutic that met safety criteria but failed efficacy checkpoints against both Alzheimer's and Huntington's diseases. It was recently found that PBT2 possessed potent antimicrobial activity, although the mechanism of bacterial cell death is unresolved. In this body of work, we show that PBT2 has multiple mechanisms of antimicrobial action, making the development of PBT2 resistance unlikely.

Microtiter Screening Reveals Oxygen-Dependent Antimicrobial Activity of Natural Products Against Mastitis-Causing Bacteria.

In this study we investigated the influence of oxygen availability on a phenotypic microtiter screen to identify new, natural product inhibitors of growth for the bovine mastitis-causing microorganisms; Streptococcus uberis, Staphylococcus aureus, and Escherichia coli. Mastitis is a common disease in dairy cattle worldwide and is a major cause of reduced milk yield and antibiotic usage in dairy herds. Prevention of bovine mastitis commonly relies on the application of teat disinfectants that contain either iodine or chlorhexidine. These compounds are used extensively in human clinical settings and increased tolerance to chlorhexidine has been reported in both Gram-positive and Gram-negative microorganisms. As such new, non-human use alternatives are required for the agricultural industry. Our screening was conducted under normoxic (20% oxygen) and hypoxic (<1% oxygen) conditions to mimic the conditions on teat skin and within the mammary gland respectively, against two natural compound libraries. No compounds inhibited E. coli under either oxygen condition. Against the Gram-positive microorganisms, 12 inhibitory compounds were identified under normoxic conditions, and 10 under hypoxic conditions. Data revealed a clear oxygen-dependency amongst compounds inhibiting growth, with only partial overlap between oxygen conditions. The oxygen-dependent inhibitory activity of a naturally occurring quinone, ß-lapachone, against S. uberis was subsequently investigated and we demonstrated that this compound is only active under normoxic conditions with a minimum inhibitory concentration and minimum bactericidal concentration of 32 µM and kills via a reactive oxygen species-dependent mechanism as has been demonstrated in other microorganisms. These results demonstrate the importance of considering oxygen-availability in high-throughput inhibitor discovery.

The Selective Rat Toxicant Norbormide Blocks KATP Channels in Smooth Muscle Cells But Not in Insulin-Secreting Cells.

Norbormide is a toxicant selective for rats to which it induces a widespread vasoconstriction. In a recent paper, we hypothesized a role of ATP-sensitive potassium (KATP) channels in norbormide-induced vasoconstriction. The current study was undertaken to verify this hypothesis by comparing the effects of norbormide with those of glibenclamide, a known KATP channel blocker. The whole-cell patch-clamp method was used to record KATP currents in myocytes freshly isolated from the rat and mouse caudal artery and from the rat gastric fundus, as well as in insulin-secreting pancreatic beta cells (INS-1 cells). Smooth muscle contractile function was assessed on either rat caudal artery rings or gastric fundus strips. Molecular modeling and docking simulation to KATP channel proteins were investigated in silico. Both norbormide (a racemic mixture of endo and exo isomers) and glibenclamide inhibited KATP currents in rat and mouse caudal artery myocytes, as well as in gastric fundus smooth muscle cells. In rat INS-1 cells, only glibenclamide blocked KATP channels, whereas norbormide was ineffective. The inhibitory effect of norbormide in rat caudal artery myocytes was not stereo-specific as both the endo isomers (active as vasoconstrictor) and the exo isomers (inactive as vasoconstrictor) had similar inhibitory activity. In rat caudal artery rings, norbormide-induced contraction was partially reverted by the KATP channel opener pinacidil. Computational approaches indicated the SUR subunit of KATP channels as the binding site for norbormide. KATP channel inhibition may play a role in norbormide-induced vasoconstriction, but does not explain the species selectivity, tissue selectivity, and stereoselectivity of its constricting activity. The lack of effect in INS-1 cells suggests a potential selectivity of norbormide for smooth muscle KATP channels.

Live applications of norbormide-based fluorescent probes in Drosophila melanogaster.

In this study we investigated the performance of two norbormide (NRB)-derived fluorescent probes, NRBMC009 (green) and NRBZLW0047 (red), on dissected, living larvae of Drosophila, to verify their potential application in live cell imaging confocal microscopy. To this end, larval tissues were exposed to NRB probes alone or in combination with other commercial dyes or GFP-tagged protein markers. Both probes were rapidly internalized by most tissues (except the central nervous system) allowing each organ in the microscope field to be readily distinguished at low magnification. At the cellular level, the probes showed a very similar distribution (except for fat bodies), defined by loss of signal in the nucleus and plasma membrane, and a preferential localization to endoplasmic reticulum (ER) and mitochondria. They also recognized ER and mitochondrial phenotypes in the skeletal muscles of fruit fly models that had loss of function mutations in the atlastin and mitofusin genes, suggesting NRBMC009 and NRBZLW0047 as potentially useful screening tools for characterizing ER and mitochondria morphological alterations. Feeding of larvae and adult Drosophilae with the NRB-derived dyes led to staining of the gut and its epithelial cells, revealing a potential role in food intake assays. In addition, when flies were exposed to either dye over their entire life cycle no apparent functional or morphological abnormalities were detected. Rapid internalization, a bright signal, a compatibility with other available fluorescent probes and GFP-tagged protein markers, and a lack of toxicity make NRBZLW0047 and, particularly, NRBMC009 highly performing fluorescent probes for live cell microscopy studies and food intake assays in Drosophila.

Very Short and Stable Lactoferricin-Derived Antimicrobial Peptides: Design Principles and Potential Uses.

The alarming rate at which micro-organisms are developing resistance to conventional antibiotics represents one of the global challenges of our time. There is currently ample space in the antibacterial drug pipeline, and scientists are trying to find innovative and novel strategies to target the microbial enemies. Nature has remained a source of inspiration for most of the antibiotics developed and used, and the immune molecules produced by the innate defense systems, as a first line of defense, have been heralded as the next source of antibiotics. Most living organisms produce an arsenal of antimicrobial peptides (AMPs) to rapidly fend off intruding pathogens, and several different attempts have been made to transform this versatile group of compounds into the next generation of antibiotics. However, faced with the many hurdles of using peptides as drugs, the success of these defense molecules as therapeutics remains to be realized. AMPs derived from the proteolytic degradation of the innate defense protein lactoferrin have been shown to display several favorable antimicrobial properties. In an attempt to investigate the biological and pharmacological properties of these much shorter AMPs, the sequence dependence was investigated, and it was shown, through a series of truncation experiments, that these AMPs in fact can be prepared as tripeptides, with improved antimicrobial activity, via the incorporation of unnatural hydrophobic residues and terminal cappings. In this Account, we describe how this class of promising cationic tripeptides has been developed to specifically address the main challenges limiting the general use of AMPs. This has been made possible through the identification of the antibacterial pharmacophore and via the incorporation of a range of unnatural hydrophobic and cationic amino acids. Incorporation of these residues at selected positions has allowed us to extensively establish how these compounds interact with the major proteolytic enzymes trypsin and chymotrypsin and also the two major drug-binding plasma proteins serum albumin and α-1 glycoprotein. Several of the challenges associated with using AMPs relate to their size, susceptibility to rapid proteolytic degradation, and poor oral bioavailability. Our studies have addressed these issues in detail, and the results have allowed us to effectively design and prepare active and metabolically stable AMPs that have been evaluated in a range of functional settings. The optimized short AMPs display inhibitory activities against a plethora of micro-organisms at low micromolar concentrations, and they have been shown to target resistant strains of both bacteria and fungi alike with a very rapid mode of action. Our Account further describes how these compounds behave in in vivo experiments and highlights both the challenges and possibilities of the intriguing compounds. In several areas, they have been shown to exhibit comparable or superior activity to established antibacterial, antifungal, and antifouling commercial products. This illustrates their ability to effectively target and eradicate various microbes in a variety of settings ranging from the ocean to the clinic.

Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging.

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12. Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells.

'Tethering' fragment-based drug discovery to identify inhibitors of the essential respiratory membrane protein type II NADH dehydrogenase.

Energy generation is a promising area of drug discovery for both bacterial pathogens and parasites. Type II NADH dehydrogenase (NDH-2), a vital respiratory membrane protein, has attracted attention as a target for the development of new antitubercular and antimalarial agents. To date, however, no potent, specific inhibitors have been identified. Here, we performed a site-directed screening technique, tethering-fragment based drug discovery, against wild-type and mutant forms of NDH-2 containing engineered active-site cysteines. Inhibitory fragments displayed IC50 values between 3 and 110 µM against NDH-2 mutants. Possible binding poses were investigated by in silico modelling, providing a basis for optimisation of fragment binding and improved potency against NDH-2.

Norborn-2-en-7-ones as physiologically-triggered carbon monoxide-releasing prodrugs.

A prodrug strategy for the release of the gasotransmitter CO at physiological pH, based upon 3a-bromo-norborn-2-en-7-one Diels-Alder cycloadducts of 2-bromomaleimides and 2,5-dimethyl-3,4-diphenylcyclopentadienone has been developed. Examples possessing protonated amine and diamine groups showed good water solubility and thermal stability. Half-lives for CO-release in TRIS-sucrose buffer at pH 7.4 ranged from 19 to 75 min at 37 °C and 31 to 32 h at 4 °C. Bioavailability in rats was demonstrated by oral gavage and oCOm-21 showed a dose dependent vasorelaxant effect in pre-contracted rat aortic rings with an EC50 of 1.6 ± 0.9 µM. Increased intracellular CO levels following oCOm-21 exposure were confirmed using a CO specific fluorescent probe.

An NBD Derivative of the Selective Rat Toxicant Norbormide as a New Probe for Living Cell Imaging.

Norbormide (NRB) is a unique compound that acts directly on rat vascular myocytes to trigger a contractile process, through an as yet unknown mechanism, which results in the selective contraction of rat peripheral arteries. To gain insight into the mechanisms involved in NRB rat-selective activity, we investigated the subcellular distribution of NRB-AF12, a nitrobenzoxadiazole (NBD)-derivative of NRB, in living NRB-sensitive and NRB-insensitive cells. In both cell types, NRB-AF12 localized to the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and endosomes; however, in NRB-sensitive cells, the fluorescence also extended to the plasma membrane. NRB-AF12 was rapidly internalized into the cells, could easily be washed out and then reloaded back into the same cells, all with a high degree of reproducibility. Cells exposed for 24 h to NRB-AF12 did not show apparent signs of toxicity, even at concentrations of the dye (10 µM) much higher than those required for fluorescence labeling (500 ηM). The distribution pattern of NRB-AF12 fluorescence was near identical to that of ER-Tracker® (Er-Tr), a fluorescent derivative of glibenclamide, a known KATP channel blocker. Displacement tests did not demonstrate, but at the same time did not rule out the possibility of a common target for ER-Tr, NRB-AF12, NRB, and glibenclamide. On the basis of these results we hypothesize a common target site for NRB-AF12 and ER-Tr, and a similar target profile for NRB and glibenclamide, and propose NRB-AF12 as an alternative fluorescence probe to ER-Tracker. Furthermore, NRB-based fluorescence derivatives could be designed to selectively label single cellular structures.