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.

Potential Targets for Antifungal Drug Discovery Based on Growth and Virulence in Candida albicans.

Fungal infections, especially infections caused by Candida albicans, remain a challenging problem in clinical settings. Despite the development of more-effective antifungal drugs, their application is limited for various reasons. Thus, alternative treatments with drugs aimed at novel targets in C. albicans are needed. Knowledge of growth and virulence in fungal cells is essential not only to understand their pathogenic mechanisms but also to identify potential antifungal targets. This article reviews the current knowledge of the mechanisms of growth and virulence in C. albicans and examines potential targets for the development of new antifungal drugs.

Itaconic Acid: The Surprising Role of an Industrial Compound as a Mammalian Antimicrobial Metabolite.

Itaconic acid is well known as a precursor for polymer synthesis and has been involved in industrial processes for decades. In a recent surprising discovery, itaconic acid was found to play a role as an immune-supportive metabolite in mammalian immune cells, where it is synthesized as an antimicrobial compound from the citric acid cycle intermediate cis-aconitic acid. Although the immune-responsive gene 1 protein (IRG1) has been associated to immune response without a mechanistic function, the critical link to itaconic acid production through an enzymatic function of this protein was only recently revealed. In this review, we highlight the history of itaconic acid as an industrial and antimicrobial compound, starting with its biotechnological synthesis and ending with its antimicrobial function in mammalian immune cells.

Non-traditional antibacterial screening approaches for the identification of novel inhibitors of the glyoxylate shunt in gram-negative pathogens.

Antibacterial compounds that affect bacterial viability have traditionally been identified, confirmed, and characterized in standard laboratory media. The historical success of identifying new antibiotics via this route has justifiably established a traditional means of screening for new antimicrobials. The emergence of multi-drug-resistant (MDR) bacterial pathogens has expedited the need for new antibiotics, though many in the industry have questioned the source(s) of these new compounds. As many pharmaceutical companies' chemical libraries have been exhaustively screened via the traditional route, we have concluded that all compounds with any antibacterial potential have been identified. While new compound libraries and platforms are being pursued, it also seems prudent to screen the libraries we currently have in hand using alternative screening approaches. One strategy involves screening under conditions that better reflect the environment pathogens experience during an infection, and identifying in vivo essential targets and pathways that are dispensable for growth in standard laboratory media in vitro. Here we describe a novel screening strategy for identifying compounds that inhibit the glyoxylate shunt in Pseudomonas aeruginosa, a pathway that is required for bacterial survival in the pulmonary environment. We demonstrate that these compounds, which were not previously identified using traditional screening approaches, have broad-spectrum antibacterial activity when they are tested under in vivo-relevant conditions. We also show that these compounds have potent activity on both enzymes that comprise the glyoxylate shunt, a feature that was supported by computational homology modeling. By dual-targeting both enzymes in this pathway, we would expect to see a reduced propensity for resistance development to these compounds. Taken together, these data suggest that understanding the in vivo environment that bacterial pathogens must tolerate, and adjusting the antibacterial screening paradigm to reflect those conditions, could identify novel antibiotics for the treatment of serious MDR pathogens.

Germination and storage reserve mobilization are regulated independently in Arabidopsis.

The phytohormone abscisic acid (ABA) inhibits the germination of many seeds, including Arabidopsis, but the mechanism for this is not known. In cereals, ABA inhibits the expression of genes involved in storage reserve mobilization. We have found that in Arabidopsis ABA decreases transcription from the promoters of marker genes for beta-oxidation and the glyoxylate cycle, essential pathways for the conversion of storage lipid (triacylglycerol) into sucrose. Thirty per cent of stored lipid is broken down over 6 days following imbibition of ABA-treated seed. Sucrose levels in ABA-treated seeds, rather than decreasing as under normal growth conditions, actually double during the 3 days following imbibition. This sucrose is derived from triacylglycerol as demonstrated by two mutants disrupted in the conversion of triacylglycerol into sucrose, kat2 and icl1, which do not accumulate sucrose in the presence of ABA. We conclude that the ABA block on germination is not a consequence of inhibition of storage lipid mobilization. Two independent programmes appear to operate, one that is blocked by ABA, governing developmental growth resulting in germination; and a second that governs storage lipid mobilization which is largely ABA-independent.

Pharmacological approaches in the treatment of primary hyperoxaluria.

Drug therapy receives scant attention as a treatment mode for primary hyperoxaluria (PH). Currently, pyridoxine is the only drug in the arsenal and only a minority of PH1 patients respond to it. In this report a pathway describing the synthesis of glyoxylate, the major precursor of oxalate, is proposed and potential drugs that may be effective in inhibiting hepatic oxalate synthesis are discussed. One of these, (L)-oxothiazolidine-4-carboxylate (OTZ), is currently undergoing evaluation in a Phase II clinical trial. It is suggested that an ideal drug may be an antisense oligonucleotide that blocks the expression of glycolate oxidase, a key enzyme in hepatic oxalate synthesis.

Some properties of serine: glyoxylate aminotransferase from rye seedlings (Secale cereale L.).

Serine: glyoxylate aminotransferase (EC 2.6.1.45) from rye seedlings catalysed transamination between L-serine and glyoxylate according to the Ping Pong Bi Bi mechanism with double substrate inhibition. As judged from the Km values, L-serine, L-alanine, and L-asparagine served as substrates for the enzyme with glyoxylate, whereas L-alanine and L-asparagine underwent transamination with hydroxypyruvate as acceptor. Pyridoxal phosphate (PLP) seems to be rather loosely bound to the enzyme protein. Aminooxyacetate and D-serine were found to be pure competitive inhibitors of the enzyme, with Ki values of 0.12 microM and 1.6 mM, respectively. Among the PLP inhibitors isonicotinic acid hydrazide and hydroxylamine were far less effective than aminooxyacetate (20% and 70% inhibition at 0.1 mM concentration, respectively). Inhibition by the SH group inhibitors at 1 mM concentration did not exceed 50%. L-Serine distinctly diminished the inhibitory effect of this type inhibitors. Preincubation of the enzyme with glyoxylate distinctly diminished transamination. Glyoxylate limited the inhibitory action of formaldehyde probably by competing for the reactive groups present in the active centre.

Purification and characterization of serine-glyoxylate aminotransferase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2.

Serine--glyoxylate aminotransferase was purified to complete homogeneity from a serine-producing methylotrophic bacterium, Hyphomicrobium methylovorum GM2, which possesses the serine pathway. This is the first microbial serine--glyoxylate aminotransferase to be purified. The enzyme has a molecular mass of about 140 kDa and consists of four subunits of identical mass, i.e. 40 kDa. The holoenzyme exhibited absorption maxima at 282 nm and 408 nm, and a shoulder at about 315-345 nm in potassium phosphate pH 7.0; it contained 4 mol pyridoxal 5'-phosphate/mol enzyme. Isoelectric focusing showed that the enzyme had a pI value of 6.9. The Km values for glyoxylate and L-serine were 0.23 mM and 4.98 mM, respectively, and the enzyme showed high specificity for these substrates. The transamination between glyoxylate and L-serine seemed to be nearly irreversible. These data indicated that this serine--glyoxylate aminotransferase plays an essential role in methanol assimilation through the serine pathway in H. methylovorum GM2.

Kinetic properties and characteristics of mouse liver mitochondrial asparagine aminotransferase.

Mouse liver asparagine aminotransferase has been found to be a mixture of enzyme forms having a cytosolic component and a mitochondrial component. The molecular weight of the mitochondrial enzyme is 70,800. The mitochondrial asparagine aminotransferase is strongly inhibited by aminooxyacetate. It is less affected by D-cycloserine but a small amount of inhibition is observed. Cysteine strongly inhibits the enzyme as do several sulfhydryl modifying reagents. The activities of the cytosolic and mitochondrial aminotransferases have been separated, and the kinetic properties of the mitochondrial form determined. The mouse liver mitochondrial asparagine aminotransferase is fairly specific for asparagine, utilizing very few amino acids as alternate amino donors and none to a great extent. The keto acid specificity is very broad, but glyoxylate is one of the most active amino group acceptors. The kinetic properties of the mitochondrial enzyme are also reported here and the data indicate strong substrate and product inhibition. Abortive complex formation may account for the deviation of the double reciprocal plots from the expected pattern.

A comparison of the cytosolic and mitochondrial forms of asparagine/glyoxylate aminotransferase.

Asparagine aminotransferase activity was measured in a variety of mouse tissues. The liver had the highest activity--nearly 20 times more than any of the other tissues tested. Hepatic asparagine aminotransferase was found to consist of cytosolic and mitochondrial forms. The mitochondrial form was found to be the predominant form in mouse tissue. Gel filtration chromatography indicated that the mouse enzyme forms have comparable molecular weights of approximately 70,000. While the substrate specificities of the two forms are very different, asparagine was the preferred amino donor for both forms. The relative contribution to the total activity of the hepatic enzyme forms varies with the animal source. Mouse had the highest level of enzyme activity of all animals tested. Ratios of the two enzyme forms also varied greatly not only with the animal source but also with the substrate used and the isolation conditions.

Identity of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase.

Identification of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase has been done. When a mitochondria extract was subjected to isoelectric focusing, the two enzyme activities were identically focused. This procedure and DEAE-Sepharose chromatography revealed multiple forms of the enzyme, in which the main form was purified. In the various purification steps the two enzyme activities appeared in the same fraction. The enzyme of the final preparation step gave a single band in polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. During the purification, a similar increase of the specific activity and yield were obtained in the two activities. Phenylalanine was found to be a competitive inhibitor of asparagine transaminase. These results suggest the identity of the two enzymes.