Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activator

BACKGROUND: Urokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents. RESULTS: 4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, K(i )= 0.034 μM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups. CONCLUSION: This study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors

Inhibition of pancreatic cholesterol esterase reduces cholesterol absorption in the hamster

BACKGROUND: Pancreatic cholesterol esterase has three proposed functions in the intestine: 1) to control the bioavailability of cholesterol from dietary cholesterol esters; 2) to contribute to incorporation of cholesterol into mixed micelles; and 3) to aid in transport of free cholesterol to the enterocyte. Inhibitors of cholesterol esterase are anticipated to limit the absorption of dietary cholesterol. RESULTS: The selective and potent cholesterol esterase inhibitor 6-chloro-3-(1-ethyl-2-cyclohexyl)-2-pyrone (figure 1, structure 1) was administered to hamsters fed a high cholesterol diet supplemented with radiolabeled cholesterol ester. Hamsters were gavage fed (3)H-labeled cholesteryl oleate along with inhibitor 1, 0–200 micromoles. Twenty-four hours later, hepatic and serum radioactive cholesterol levels were determined. The ED(50 )of inhibitor 1 for prevention of the uptake of labeled cholesterol derived from hydrolysis of labeled cholesteryl oleate was 100 micromoles. The toxicity of inhibitor 1 was investigated in a 30 day feeding trial. Inhibitor 1, 100 micromoles or 200 micromoles per day, was added to chow supplemented with 1% cholesterol and 0.5% cholic acid. Clinical chemistry urinalysis and tissue histopathology were obtained. No toxicity differences were noted between control and inhibitor supplemented groups. CONCLUSIONS: Inhibitors of cholesterol esterase may be useful therapeutics for limiting cholesterol absorption

La ionosfera: comunicare... naturalmente!

La ionosfera è la parte della media-alta atmosfera compresa tra i 60 e i 1000 km di quota. Essa è caratterizzata da una concentrazione di elettroni tale da modificare la propagazione delle onde radio che la attraversano

Effects of Surface Geology on Seismic Ground Motion Deduced from Ambient-Noise Measurements in the Town of Avellino, Irpinia Region (Italy)

The effects of surface geology on ground motion provide an important tool in seismic hazard studies. It is well known that the presence of soft sediments can cause amplification of the ground motion at the surface, particularly when there is a sharp impedance contrast at shallow depth. The town of Avellino is located in an area characterised by high seismicity in Italy, about 30 km from the epicentre of the 23 November 1980, Irpinia earthquake (M = 6.9). No earthquake recordings are available in the area. The local geology is characterised by strong heterogeneity, with impedance contrasts at depth. We present the results from seismic noise measurements carried out in the urban area of Avellino to evaluate the effects of local geology on the seismic ground motion. We computed the horizontal-to-vertical (H/V) noise spectral ratios at 16 selected sites in this urban area for which drilling data are available within the first 40 m of depth. A Rayleigh wave inversion technique using the peak frequencies of the noise H/V spectral ratios is then presented for estimating Vs models, assuming that the thicknesses of the shallow soil layers are known. The results show a good correspondence between experimental and theoretical peak frequencies, which are interpreted in terms of sediment resonance. For one site, which is characterised by a broad peak in the horizontal-to-vertical spectral-ratio curve, simple one-dimensional modelling is not representative of the resonance effects. Consistent variations in peak amplitudes are seen among the sites. A site classification based on shear-wave velocity characteristics, in terms of Vs30, cannot explain these data. The differences observed are better correlated to the impedance contrast between the sediments and basement. A more detailed investigation of the physical parameters of the subsoil structure, together with earthquake data, are desirable for future research, to confirm these data in terms of site response

Reductive Metabolism of AGE Precursors: A Metabolic Route for Preventing AGE Accumulation in Cardiovascular Tissue

OBJECTIVE—To examine the role of aldo-keto reductases (AKRs) in the cardiovascular metabolism of the precursors of advanced glycation end products (AGEs). RESEARCH DESIGN AND METHODS—Steady-state kinetic parameters of AKRs with AGE precursors were determined using recombinant proteins expressed in bacteria. Metabolism of meth-ylglyoxal and AGE accumulation were studied in human umbil-ical vein endothelial cells (HUVECs) and C57 wild-type, akr1b3 (aldose reductase)-null, cardiospecific-akr1b4 (rat aldose reduc-tase), and akr1b8 (FR-1)-transgenic mice. AGE accumulation and atherosclerotic lesions were studied 12 weeks after streptozoto-cin treatment of C57, akr1b3-null, and apoE- and akr1b3-apoE– null mice. RESULTS—Higher levels of AGEs were generated in the cytosol than at the external surface of HUVECs cultured in high glucose

Expression of mitochondrial protein genes encoded by nuclear and mitochondrial genomes correlate with energy metabolism in dairy cattle

Background Mutations in the mitochondrial genome have been implicated in mitochondrial disease, often characterized by impaired cellular energy metabolism. Cellular energy metabolism in mitochondria involves mitochondrial proteins (MP) from both the nuclear (NuMP) and mitochondrial (MtMP) genomes. The expression of MP genes in tissues may be tissue specific to meet varying specific energy demands across the tissues. Currently, the characteristics of MP gene expression in tissues of dairy cattle are not well understood. In this study, we profile the expression of MP genes in 29 adult and six foetal tissues in dairy cattle using RNA sequencing and gene expression analyses: particularly differential gene expression and co-expression network analyses. Results MP genes were differentially expressed (DE; over-expressed or under-expressed) across tissues in cattle. All 29 tissues showed DE NuMP genes in varying proportions of over-expression and under-expression. On the other hand, DE of MtMP genes was observed in < 50% of tissues and notably MtMP genes within a tissue was either all over-expressed or all under-expressed. A high proportion of NuMP (up to 60%) and MtMP (up to 100%) genes were over-expressed in tissues with expected high metabolic demand; heart, skeletal muscles and tongue, and under-expressed (up to 45% of NuMP, 77% of MtMP genes) in tissues with expected low metabolic rates; leukocytes, thymus, and lymph nodes. These tissues also invariably had the expression of all MtMP genes in the direction of dominant NuMP genes expression. The NuMP and MtMP genes were highly co-expressed across tissues and co-expression of genes in a cluster were non-random and functionally enriched for energy generation pathway. The differential gene expression and co-expression patterns were validated in independent cow and sheep datasets. Conclusions The results of this study support the concept that there are biological interaction of MP genes from the mitochondrial and nuclear genomes given their over-expression in tissues with high energy demand and co-expression in tissues. This highlights the importance of considering MP genes from both genomes in future studies related to mitochondrial functions and traits related to energy metabolism

Overexpression of Leucyl Aminopeptidase in Plasmodium falciparum Parasites. Target for the antimalarial activity of bestatin

Malaria aminopeptidases are important in the generation and regulation of free amino acids that are used in protein anabolism and for maintaining osmotic stability within the infected erythrocyte. The intraerythrocytic development of malaria parasites is blocked when the activity of aminopeptidases is specifically inhibited by reagents such as bestatin. One of the major aminopeptidases of malaria parasites is a leucyl aminopeptidase of the M17 family. We reasoned that, when this enzyme was the target of bestatin inhibition, its overexpression in malaria cells would lead to a reduced sensitivity to the inhibitor. To address this supposition, transgenic Plasmodium falciparum parasites overexpressing the leucyl aminopeptidase were generated by transfection with a plasmid that housed the full-length gene. Transgenic parasites expressed a 65-kDa protein close to the predicted molecule size of 67.831 kDa for the introduced leucyl aminopeptidase, and immunofluorescence studies localized the protein to the cytosol, the location of the native enzyme. The product of the transgene was shown to be functionally active with cytosolic extracts of transgenic parasites exhibiting twice the leucyl aminopeptidase activity compared with wildtype parasites. In vitro inhibitor sensitivity assays demonstrated that the transgenic parasites were more resistant to bestatin (EC50 64 mu M) compared with the parent parasites (EC50 25 mu M). Overexpression of genes in malaria parasites would have general application in the identification and validation of targets for antimalarial drugs