Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson's disease.

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

Daphnane Diterpenes from Daphne genkwa Activate Nurr1 and Have a Neuroprotective Effect in an Animal Model of Parkinson's Disease.

Nurr1 is an orphan nuclear receptor that is essential for the differentiation and maintenance of dopaminergic neurons in the brain, and it is a therapeutic target for Parkinson's disease (PD). During the screening for Nurr1 activators from natural sources using cell-based assay systems, a methanol extract of the combined stems and roots of Daphne genkwa was found to activate the transcriptional function of Nurr1 at a concentration of 3 µg/mL. The active components were isolated and identified as genkwanine N (1) and yuanhuacin (2). Both compounds 1 and 2 significantly enhanced the function of Nurr1 at 0.3 µM. Nurr1-specific siRNA abolished the activity of 1 and 2, strongly suggesting that transcriptional activation by 1 and 2 occurred through the modulation of Nurr1 function. Additionally, treatment with 1 and 2 inhibited 6-hydroxydopamine (6-OHDA)-induced neuronal cell death and lipopolysaccharide (LPS)-induced neuroinflammation. Moreover, in a 6-OHDA-lesioned rat model of PD, intraperitoneal administration of 2 (0.5 mg/kg/day) for 2 weeks significantly improved behavioral deficits and reduced tyrosine hydroxylase (TH)-positive dopaminergic neuron death induced by 6-OHDA injection and had a beneficial effect on the inflammatory response in the brain. Accordingly, compounds 1 and 2, the first reported Nurr1 activators of natural origin, are potential lead compounds for the treatment of PD.

Verrulactone C with an unprecedented dispiro skeleton, a new inhibitor of Staphylococcus aureus enoyl-ACP reductase, from Penicillium verruculosum F375.

An highly quaternary and unprecedented dispiro compound, verrulactone C, with the known compound, altenuisol, were isolated from a culture broth of the fungal strain Penicillium verruculosum F375 and their structures were established by various spectral analysis. Verrulactone C and altenuisol showed FabI-selective inhibition. Especially altenuisol had the high correlation between FabI-inhibition and whole cell antibacterial activity against Staphylococcus aureus and MRSA with MICs of 8-32µg/mL.

The Lactone form of stachybotrydial: a new inhibitor of dihydrofolate reductase from stachybotrys sp. FN298.

Dihydrofolate reductase (DHFR) has been confirmed to be a novel target for antibacterial drug development. In this study, we determined that a fungal metabolite from Stachybotrys sp. FN298 can inhibit the DHFR of Staphylococcus aureus. Its structure was identified as a lactone form of stachybotrydial using mass spectrometry and nuclear magnetic resonance analysis. This compound inhibited S. aureus DHFR with a half-maximal inhibitory concentration of 41 µM. It also prevented the growth of S. aureus and methicillin-resistant S. aureus (MRSA) with a minimum inhibitory concentration of 32 µg·mL(-1). To our knowledge, this is the first description of a DHFR inhibitor of microbial origin. The inhibitory function of the lactone form of stachybotrydial highlights its potential for development into a new broad-spectrum antibacterial agent and as an agent against MRSA.

Verrulactones A and B, new inhibitors of Staphylococcus aureus enoyl-ACP reductase produced by Penicillium verruculosum F375.

New dimeric compounds of alternariol class, verrulactones A and B, were isolated from a culture broth of the fungal strain Penicillium verruculosum F375 and their structure were established by various spectral analysis. Verrulactones A and B strongly inhibited Staphylococcus aureus enoyl-ACP reductase with IC(50) of 0.92 and 1.41 µM, respectively, and also showed antibacterial activity against S. aureus and MRSA with MICs of 8 and 16 µg/mL, respectively.

Flavimycins A and B, dimeric 1,3-dihydroisobenzofurans with peptide deformylase inhibitory activity from Aspergillus flavipes.

Flavimycins A (1) and B (2), novel dimeric 1,3-dihydroisobenzofurans, were isolated as inhibitors of peptide deformylase from cultures of Aspergillus flavipes. Their chemical structures were established by NMR and MS data analysis. Compounds 1 and 2 exist as epimeric mixtures at C-1 through fast hemiacetal-aldehyde tautomerism. Compounds 1 and 2 inhibited Staphylococcus aureus peptide deformylase with IC50 values of 35.8 and 100.1 µM, respectively. Consistent with their PDF inhibition, 1 showed two times stronger antibacterial activity than 2 on S. aureus including MRSA, with MIC values of 32-64 µg/mL.

Chalcomoracin and moracin C, new inhibitors of Staphylococcus aureus enoyl-acyl carrier protein reductase from Morus alba.

Bacterial enoyl-acyl carrier protein (ACP) reductase has been confirmed as a novel target for antibacterial drug development. In the screening of inhibitors of Staphylococcus aureus enoyl-ACP reductase (FabI), we found that a methanol extract of leaves of Morus alba L. potently inhibited S. aureus FabI as well as growth of S. aureus. The active principles were identified as chalcomoracin and moracin C by MS and NMR analysis. Chalcomoracin and moracin C inhibited S. aureus FabI with IC(50) of 5.5 and 83.8 µM, respectively. They also prevented the growth of S. aureus with minimum inhibitory concentration (MIC) of 4 and 32 µg/mL, respectively. Consistent with their inhibition against FabI and bacterial growth, they prevented (14)C]acetate incorporation into fatty acid in S. aureus while didn't affect protein synthesis. In this study, we reported that chalcomoracin and moracin C, potent antibacterial compounds from Morus alba, inhibited FabI and fatty acid synthesis.

Phellinstatin, a new inhibitor of enoyl-ACP reductase produced by the medicinal fungus Phellinus linteus.

A new trimeric hispidin derivative, phellinstatin, was isolated from a culture broth of the medicinal fungus Phellinus linteus and its structure was established by various spectral analysis. Phellinstatin strongly inhibited Staphylococcus aureus enoyl-ACP reductase with an IC(50) of 6 µM and also showed antibacterial activity against S. aureus and MRSA.

Methyl-branched fatty acids, inhibitors of enoyl-ACP reductase with antibacterial activity from Streptomyces sp. A251.

Bacterial enoyl-ACP reductase (FabI) has been demonstrated to be a novel antibacterial target. In the course of our screening for FabI inhibitors we isolated two methyl-branched fatty acids from Streptomyces sp. A251. They were identified as 14-methyl-9(Z)-pentadecenoic acid and 15-methyl-9(Z)-hexadecenoic acid by MS and NMR spectral data. These compounds inhibited Staphylococcus aureus FabI with IC50 of 16.0 and 16.3mu M, respectively, while didn't affect FabK, an enoyl-ACP reductase of Streptococcus pneumonia, at 100muM. Consistent with their selective inhibition for FabI, they blocked intracellular fatty acid synthesis as well as the growth of S. aureus, while didn't inhibit the growth of S. pneumonia. Additionally, these compounds showed reduced antibacterial activity against fabI-overexpressing S. aureus compared to the wild-type strain. These results demonstrate that the methyl-branched fatty acids showed antibacterial activity by inhibiting FabI in vivo.

Vinaxanthone, a new FabI inhibitor from Penicillium sp.

OBJECTIVES: Bacterial enoyl-ACP reductase (FabI) has been validated as a novel antibacterial target for tackling infections caused by multidrug-resistant pathogens. A few FabI inhibitors, however, are known. This study isolated a new FabI inhibitor from Penicillium sp. METHODS: A screening programme led to the selection of a Penicillium sp. producing a strong FabI-inhibitory metabolite. The chemical structure of the isolated FabI inhibitor was elucidated by mass spectrometry and nuclear magnetic resonance spectral data. The antibacterial target of the inhibitor was validated by overexpression assays. RESULTS: The isolated FabI inhibitor was elucidated to be vinaxanthone. It selectively inhibited Staphylococcus aureus FabI with an IC(50) of 0.9 microM; it did not affect FabK, an enoyl-ACP reductase of Streptococcus pneumoniae. Consistent with its inhibition of FabI, the inhibitor prevented intracellular fatty acid synthesis while it did not affect protein biosynthesis. It also prevented the growth of S. aureus as well as methicillin-resistant S. aureus (MRSA) and quinolone-resistant S. aureus. Importantly, fabI-overexpressing S. aureus showed reduced susceptibility to the inhibitor compared with the wild-type strain, demonstrating that its antibacterial action is mediated by inhibition of FabI. CONCLUSIONS: Vinaxanthone is a new FabI-directed antibacterial of natural origin that could have potential for further development as a new anti-MRSA agent.

Macrolactin S, a new antibacterial agent with FabG-inhibitory activity from Bacillus sp. AT28.

In the course of screening for FabG inhibitors from microbial sources, a new 24-membered ring lactone named macrolactin S, along with the known compound macrolactin B, has been isolated from the mycelium of liquid fermentation cultures of Bacillus sp. AT28. The structure of macrolactin S was determined on the basis of MS and NMR data. Macrolactin S showed a dose-dependent inhibition of Staphylococcus aureus FabG, not inhibiting S. aureus FabI. Also macrolactin S inhibited the growth of S. aureus, Bacillus subtilis, and Escherichia coli.

Naturally acquired IgM antibody response to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax in Korea: use for serodiagnosis of vivax malaria.

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-naïve healthy individuals (control group 1), and 70 malaria-naïve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.

Fumimycin: a peptide deformylase inhibitor with an unusual skeleton produced by Aspergillus fumisynnematus.

Fumimycin, an unusual metabolite incorporating an unusual alanine unit linked to a phenyl group at the alpha-carbon with both lactone and amide moieties, was isolated from cultures of Aspergillus fumisynnematus. Its structure was established by spectral analysis. Fumimycin was found to inhibit Staphylococcus aureus peptide deformylase with an IC50 value of 4.1 microM and also showed antibacterial activity against S. aureus.

Protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in rat cortical neuron-glia cultures.

We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron-glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-gamma-induced neurotoxicity in rat cortical neuron-glia cultures with an EC(50) value of 0.09 microM. Radicicol inhibited the LPS/IFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-gamma-induced release of tumor necrosis factor-alpha (TNF-alpha) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-alpha was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in neuron-glia cultures is mediated via the inhibition of TNF-alpha release, as well as the suppression of iNOS expression in microglia.

Cyclo(Dehydrohistidyl-L-tryptophyl), an inhibitor of nitric oxide production from a fungal strain, Fb956.

In the course of screening for nitric oxide inhibitors in activated microglial BV-2 cells, cyclo(dehydrohistidyl-Ltryptophyl) was isolated from solid-state fermentation cultures of an unidentified fungal strain, Fb956. Its structure was determined by spectroscopic methods including 2D NMR and chiral TLC analyses. Cyclo(dehydrohistidyl-L-tryptophyl) was found to have an inhibitory activity on nitric oxide production with an IC50 of 6.5 muM in activated BV-2 cells. The structure determination and biological activity of cyclo(dehydrohistidyl- L-tryptophyl) was reported for the first time in this study.

Evaluation of a new fourth generation enzyme-linked immunosorbent assay, the LG HIV Ag-Ab Plus, with a combined HIV p24 antigen and anti-HIV-1/2/O screening test.

The LG HIV Ag-Ab Plus, a new fourth generation diagnostic assay for HIV infection, was evaluated in comparison to the Enzygnost HIV Integral, an established fourth generation HIV assay. The LG assay showed 100% sensitivity with 109 samples with anti-HIV-1, anti-HIV-2 or anti-HIV-1 group O reactivity. It also detected correctly all 51 positives on three BBI performance panels, slightly outperforming the Enzygnost HIV Integral, which detected 50. The specificity of the LG HIV Ag-Ab Plus was 99.9% with 999 sera from healthy blood donors, which was slightly inferior to the performance of the Enzygnost HIV Integral, which had 100% specificity. The LG assay showed 100% specificity with 81 specimens with underlying diseases including hepatitis B, demonstrating a low risk of cross-reactivity with other infections. The reduction of the diagnostic window by the LG HIV Ag-Ab Plus, compared to a third generation HIV assay, was 6.3 days. The LG assay also showed sufficiently high intra-person and inter-person reproducibility. The overall performance of this new fourth generation HIV assay was adequate for screening and diagnosis of HIV infection.

Atromentin and leucomelone, the first inhibitors specific to enoyl-ACP reductase (FabK) of Streptococcus pneumoniae.

Two potent inhibitors of FabK, the enoyl-acyl carrier protein (ACP) reductase of Streptococcus pneumoniae, were isolated from the solid state fermentation of an unidentified fungus F010248. Their structures were identified to be atromentin and leucomelone by various spectral analysis. Atromentin and leucomelone inhibited the FabK with IC50 values of 0.24 and 1.57 microM, respectively, while did not inhibit FabI, the enoyl-ACP reductase of either Escherichia coli or Staphylococcus aureus, even at 200 microM. Atromentin and leucomelone are the first inhibitors specific to the enoyl-ACP reductase (FabK) of Streptococcus pneumoniae.

Meleagrin, a new FabI inhibitor from Penicillium chryosogenum with at least one additional mode of action.

Bacterial enoyl-acyl carrier protein reductase (FabI) is a promising novel antibacterial target. We isolated a new class of FabI inhibitor from Penicillium chrysogenum, which produces various antibiotics, the mechanisms of some of them are unknown. The isolated FabI inhibitor was determined to be meleagrin by mass spectroscopy and nuclear magnetic resonance spectral analyses, and its more active and inactive derivatives were chemically prepared. Consistent with their selective inhibition of Staphylococcus aureus FabI, meleagrin and its more active derivatives directly bound to S. aureus FabI in a fluorescence quenching assay, inhibited intracellular fatty acid biosynthesis and growth of S. aureus, and increased the minimum inhibitory concentration for fabI-overexpressing S. aureus. The compounds that were not effective against the FabK isoform, however, inhibited the growth of Streptococcus pneumoniae that contained only the FabK isoform. Additionally no resistant mutant to the compounds was obtained. Importantly, fabK-overexpressing Escherichia coli was not resistant to these compounds, but was resistant to triclosan. These results demonstrate that the compounds inhibited another target in addition to FabI. Thus, meleagrin is a new class of FabI inhibitor with at least one additional mode of action that could have potential for treating multidrug-resistant bacteria.

Panosialins, inhibitors of enoyl-ACP reductase from Streptomyces sp. AN1761.

In the continued search for inhibitors of enoyl-acyl carrier protein (ACP) reductase, we found that four acylbenzenediol sulfate metabolites from Streptomyces sp. AN1761 potently inhibited bacterial enoyl-ACP reductases of Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. Their structures were identified as panosialins A, B, wA, and wB by MS and NMR data. They showed stronger inhibition against S. aureus FabI and S. pneumoniae FabK with IC50 of 3-5 microM than M. tuberculosis InhA with IC50 of 9-12 microM. They also exhibited a stronger antibacterial spectrum on S. aureus and S. pneumoniae than M. tuberculosis. In addition, the higher inhibitory activity of panosialin wB than panosialin B on fatty acid biosynthesis was consistent with that on bacterial growth, suggesting that they could exert their antibacterial activity by inhibiting fatty acid synthesis.