Inhibition of matrix metalloproteinase expression and cellular invasion by NF-κB inhibitors of microbial origin.

Matrix metalloproteinases (MMPs) are zinc-dependent extracellular matrix remodeling endopeptidases. MMPs cleave various matrix proteins such as collagen, elastin, gelatin and casein. MMPs are often implicated in pathological processes, such as cancer progression including metastasis. Meanwhile, microorganisms produce various secondary metabolites having unique structures. We designed and synthesized dehydroxymethylepoxyquinomicin (DHMEQ) based on the structure of epoxyquinomicin C derived from Amycolatopsis as an inhibitor of NF-κB. This compound inhibited cancer cell migration and invasion. Since DHMEQ is comparatively unstable in the body, we designed and synthesized a stable DHMEQ analog, SEMBL. SEMBL also inhibited cancer cell migration and invasion. We also looked for inhibitors of cancer cell migration and invasion from microbial culture filtrates. As a result, we isolated a known compound, ketomycin, from Actinomycetes. DHMEQ, SEMBL, and ketomycin are all NF-κB inhibitors, and inhibited the expression of MMPs in the inhibition of cellular migration and invasion. These are all compounds with comparatively low toxicity, and may be useful for the development of anti-metastasis agents.

N-acetyl cysteine restores brain glutathione loss in combined 2-cyclohexene-1-one and d-amphetamine-treated rats: relevance to schizophrenia and bipolar disorder.

Oxidative stress and reduced brain levels of glutathione have been implicated in schizophrenia and bipolar disorder. N-acetyl cysteine (NAC) is a precursor of glutathione and has additional effects on glutamate neurotransmission, neurogenesis and inflammation. While NAC treatment has shown benefits in both schizophrenia and bipolar disorder, the mechanisms of action are largely unknown. Similarly, the interaction between oxidative stress and altered dopaminergic activities in psychiatric illness is not yet characterized. This study investigated the capacity of NAC in restoring brain glutathione depletion in rats that received 2-cyclohexene-1-one (CHX, 75 mg/kg), d-amphetamine (2.5mg/kg) or both. CHX, but not amphetamine, induced significant depletion of glutathione levels in the striatum and frontal cortex. Glutathione depletion was reversed by NAC (1000 mg/kg) in saline-treated and amphetamine-treated (frontal cortex only) rats. While NAC was shown to be beneficial in this model, the lack of additional glutathione depletion by amphetamine in combination with CHX does not support a summative interaction between oxidative stress and altered dopamine transmission.

In vitro inhibition of human neutrophil histotoxicity by ambroxol: evidence for a multistep mechanism.

Neutrophils are major culprits for the protease/antiprotease imbalance during various lung diseases, that is, chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis and adult respiratory distress syndrome. Thus, these cells are presently considered an ideal target for the pharmacologic control of tissue injury during these diseases. This study was planned in order to investigate if ambroxol and its precursor bromhexine are actually capable of preventing alpha-1-antitrypsin (A1AT) inactivation by stimulated neutrophils and possibly to look into the mechanisms underlying this event. Ambroxol inhibited the production of superoxide anion by activated neutrophils, whereas bromhexine had no inhibitory effect. Ambroxol decreased the production of hypochlorous acid (HOCl) from activated neutrophils with high efficiency, whereas bromhexine had a modest activity. Ambroxol and bromhexine were capable of limiting the chlorination of monochlorodimedon by HOCl, displaying the capacity of directly scavenging the oxidant. Ambroxol decreased the release of elastase and myeloperoxidase from activated neutrophils, whereas bromhexine was ineffective. Ambroxol prevented the A1AT inactivation by neutrophils, whereas bromhexine was completely ineffective. Among drugs currently available for in vivo use in humans, ambroxol is unique by virtue of its ability to prevent neutrophil-mediated A1AT inactivation via inhibition of HOCl production as well as HOCl scavenging. Also taking into account its capacity for curbing elastase release, the drug displays the potential to lessen the burden of oxidants/proteases and to increase the antiprotease shield at the site of inflammation. Thus, ambroxol appears to be a good candidate for raising attempts to develop new therapeutic histoprotective approaches to inflammatory bronchopulmonary diseases.

Pharmacokinetics and pharmacodynamics of NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) and mesotrione, inhibitors of 4-hydroxyphenyl pyruvate dioxygenase (HPPD) following a single dose to healthy male volunteers.

AIMS: NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) and mesotrione (2-(4-methylsulphonyl-2-nitrobenzoyl)-1,3-cyclohexanedione) are inhibitors of 4-hydroxyphenyl pyruvate dioxygenase (HPPD). NTBC has been successfully used as a treatment for hereditary tyrosinaemia type 1 (HT-1), while mesotrione has been developed as an herbicide. The pharmacokinetics of the two compounds were investigated in healthy male volunteers following single oral administration. The aim of the NTBC study was to assess the bioequivalence of two different formulations and to determine the extent of the induced tyrosinaemia. The mesotrione study was performed to determine the magnitude and duration of the effect on tyrosine catabolism. Additionally, the urinary excretion of unchanged mesotrione was measured to assess the importance of this route of clearance and to help develop a strategy for monitoring occupational exposure. METHODS: A total of 28 volunteers participated in two separate studies with the compounds. In the first study, the relative bioavailability of NTBC from liquid and capsule formulations was compared and the effect on plasma tyrosine concentrations measured. In the second study the pharmacokinetics of mesotrione were determined at three doses. Plasma tyrosine concentrations were monitored and the urinary excretion of mesotrione and tyrosine metabolites was measured. RESULTS: Both compounds were well tolerated at the dose levels studied. Peak plasma concentrations of NTBC were rapidly attained following a single oral dose of 1 mg x kg(-1) body weight of either formulation and the half-life in plasma was approximately 54 h. There were no statistical differences in mean (+/- s.d.) AUC(0,infinity) (capsule 602 +/- 154 vs solution 602 +/- 146 microg x ml(-1) h) or t1/2 (capsule 55 +/- 13 vs solution 54 +/- 8 h) and these parameters supported the bioequivalence of the two formulations. Mesotrione was also rapidly absorbed, with a significant proportion of the dose eliminated unchanged in urine. The plasma half-life was approximately 1 h and was independent of dose and AUC(0,infinity) and Cmax increased linearly with dose. Following administration of 1 mg NTBC x kg(-1) in either formulation, the concentrations of tyrosine in plasma increased to approximately 1100 nmol x ml(-1). Concentrations were still approximately 8 times those of background at 14 days after dosing, but had returned to background levels within 2 months of the second dose. Administration of mesotrione resulted in an increase in tyrosine concentrations which reached a maximum of approximately 300 nmol x ml(-1) following a dose of 4 mg x kg(-1) body weight. Concentrations returned to those of background within 2 days of dosing. Urinary excretion of tyrosine metabolites was increased during the 24 h immediately following a dose of 4 mg mesotrione x kg(-1), but returned to background levels during the following 24 h period. CONCLUSIONS: NTBC and mesotrione are both inhibitors of HPPD, although the magnitude and duration of their effect on tyrosine concentrations are very different. When normalized for dose, the extent of the induced tyrosinaemia after administration of NTBC and over the duration of these studies, was approximately 400 fold greater than that following administration of mesotrione. The persistent and significant effect on HPPD following administration of NTBC make it suitable for the treatment of patients with hereditary tyrosinaemia type 1 (HT-1), whilst the minimal and transient effects of mesotrione minimize the likelihood of a clinical effect in the event of systemic exposure occurring during occupational use.

Selective protein-protein interactions direct channeling of intermediates between polyketide synthase modules.

Polyketide synthases (PKSs) have represented fertile targets for rational manipulation via protein engineering ever since their modular architecture was first recognized. However, the mechanistic principles by which biosynthetic intermediates are sequentially channeled between modules remain poorly understood. Here we demonstrate the importance of complementarity in a remarkably simple, repetitive structural motif within these megasynthases that has been implicated to affect intermodular chain transfer [Gokhale, R. S., et al. (1999) Science 284, 482]. The C- and N-terminal ends of adjacent PKS polypeptides are capped by short peptides of 20-40 residues. Mismatched sequences abolish intermodular chain transfer without affecting the activity of individual modules, whereas matched sequences can facilitate the channeling of intermediates between ordinarily nonconsecutive modules. Thus, in addition to substrate-PKS interactions and domain-domain interactions, these short interpolypeptide sequences represent a third determinant of selective chain transfer that must be taken into consideration in the protein engineering of PKSs. Preliminary biophysical studies on synthetic peptide mimics of these linkers suggest that they may adopt coiled-coil conformations.

Antimicrobial activities and antagonists of bacilysin and anticapsin.

The dipeptide antibiotic bacilysin is active against a wide range of bacteria and against Candida albicans. Its C-terminal amino acid, anticapsin, is a very poor antibacterial agent. The activities of both substances are strongly dependent on the nature of the culture medium. In a minimal medium the minimum inhibitory concentration for bacilysin with E. coli B is 10(-3) mug ml(-1). The action of bacilysin amino acids. With several bacteria, bacilysin-resistant mutants are found in unusually large numbers. It is suggested that peptide and amino acid transport systems play a role in these phenomena. The antimicrobial action of bacilysin is also inhibited by glucosamine and N-acetylglucosamine. This antibiotic may therefore interfere with glucosamine synthesis and thus with the synthesis of microbial cell walls.