Discovery of a new class of potent prolylcarboxypeptidase inhibitors derived from alanine

Efforts to modify the central proline portion of lead compound 4 lead to the discovery of novel prolylcarboxypeptidase (PrCP) inhibitors. Especially, replacement with alanine afforded compound 19 displaying more potent human and mouse PrCP inhibitory activity than 4 and an overall comparable profile.     

Discovery of benzodihydroisofurans as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase inhibitors

A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure-activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.     

Discovery of aminoheterocycles as potent and brain penetrant prolylcarboxypeptidase inhibitors

Efforts were dedicated to develop potent and brain penetrant prolylcarboxypeptidase (PrCP) inhibitors by replacing the amide group of original leads 1 and 2 with heterocycles. Aminopyrimidines including compound 32a were identified to display good PrCP inhibitory activity (32a, IC(50)=43 nM) and impressive ability to penetrate brain in mice (brain/plasma ratio: 1.4).     

A new class of prolylcarboxypeptidase inhibitors, part 1: discovery and evaluation

A new structural class of potent prolylcarboxypeptidase (PrCP) inhibitors was discovered by high-throughput screening. The series possesses a tractable SAR profile with sub-nanomolar in vitro IC(50) values. Compared to prior inhibitors, the new series demonstrated minimal activity shifts in pure plasma and complete ex vivo plasma target engagement in mouse plasma at the 20 h post-dose time point (po). In addition, the in vivo level of CNS and non-CNS drug exposure was measured.     

Mouse Shh is required for prechordal plate maintenance during brain and craniofacial morphogenesis

In humans, holoprosencephaly (HPE) is a common birth defect characterized by the absence of midline cells from brain, facial, and oral structures. To understand the pathoetiology of HPE, we investigated the involvement of mammalian prechordal plate (PrCP) cells in HPE pathogenesis and the requirement of the secreted protein sonic hedgehog (Shh) in PrCP development. We show using rat PrCP lesion experiments and DiI labeling that PrCP cells are essential for midline development of the forebrain, foregut endoderm, and ventral cranial mesoderm in mammals. We demonstrate that PrCP cells do not develop into ventral cranial mesoderm in Shh^−/− embryos. Using Shh^−/− and chimeric embryos we show that Shh signal is required for the maintenance of PrCP cells in a non-cell autonomous manner. In addition, the hedgehog (HH)-responding cells that normally appear during PrCP development to contribute to midline tissues, do not develop in the absence of Shh signaling. This suggests that Shh protein secreted from PrCP cells induces the differentiation of HH-responding cells into midline cells. In the present study, we show that the maintenance of a viable population of PrCP cells by Shh signal is an essential process in development of the midline of the brain and craniofacial structures. These findings provide new insight into the mechanism underlying HPE pathoetiology during dynamic brain and craniofacial morphogenesis.     

Discovery and optimization of orally active cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors

The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.     

Discovery of benzimidazole pyrrolidinyl amides as prolylcarboxypeptidase inhibitors

A series of benzimidazole pyrrolidinyl amides containing a piperidinyl group were discovered as novel prolylcarboxypeptidase (PrCP) inhibitors. Low-nanomolar IC₅₀’s were achieved for several analogs, of which compound 9b displayed modest ex vivo target engagement in eDIO mouse plasma. Compound 9b was also studied in vivo for its effect on weight loss and food intake in an eDIO mouse model and the results will be discussed.     

The discovery of non-benzimidazole and brain-penetrant prolylcarboxypeptidase inhibitors

Novel prolylcarboxypeptidase (PrCP) inhibitors with nanomolar IC(50) values were prepared by replacing the previously described dichlorobenzimidazole-substituted pyrrolidine amides with a variety of substituted benzylamine amides. In contrast to prior series, the compounds demonstrated minimal inhibition shift in whole serum and minimal recognition by P-glycoprotein (P-gp) efflux transporters. The compounds were also cell permeable and demonstrated in vivo brain exposure. The in vivo effect of compound (S)-6e on weight loss in an established diet-induced obesity (eDIO) mouse model was studied.     

Pyrazoles as non-classical bioisosteres in prolylcarboxypeptidase (PrCP) inhibitors

Bioisosteres are integral components of modern pharmaceutical research that allow structural optimization to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacodynamic, pharmacokinetic and physicochemical properties. A recent medicinal chemistry campaign focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investigation into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric replacement by incorporating subsequent structural modifications to maintain potency against the selected target. A heuristic model for an embedded pharmacophore is also described.     

The mammalian twisted gastrulation gene functions in foregut and craniofacial development

Extracellular modulators of cell-cell signaling control numerous aspects of organismal development. The Twisted gastrulation (Twsg1) gene product is a small, secreted cysteine-rich protein that has the unusual property of being able to either enhance or inhibit signaling by the bone morphogenetic protein (BMP) subfamily of TGF-beta type factors in a context-dependent manner. In this report, we characterize the early embryonic and skeletal phenotypes associated with loss of Twsg1 function in mice. All Twsg1 mutant mice, irrespective of genetic background, exhibit deletions of neural arches in the cervical vertebrae. In a C57BL/6 background, we also observe pronounced forebrain defects including rostral truncations, holoprosencephaly, cyclopia, as well as alterations in the first branchial arch (BA1) leading to lack of jaw (agnathia). Characterization of marker expression suggests that these defects are attributable to loss of signaling from forebrain-organizing centers including Fgf8 from the anterior neural ridge (ANR) and Shh from the prechordal plate (PrCP). In addition, we find defects in the foregut endoderm and a reduction in Hex expression, which may contribute to both the forebrain and BA1 defects.     

eNOS genotype-dependent correlation between whole blood lead and plasma nitric oxide products concentrations.

Experimental data indicate that lead exposure decreases nitric oxide (NO) availability. However, no previous study has examined whether lead exposure affects plasma nitrite/nitrate (NO(x)) concentrations in humans. In addition, the T(-786)C polymorphism affects endothelial NO synthase (eNOS) expression and endogenous NO release. Here, we investigated whether there is an association between the circulating concentrations of NO(x) and the concentrations of lead in whole blood (B-Pb) and in plasma (P-Pb) from lead-exposed subjects. In addition, we also evaluated whether eNOS genotype for the T(-786)C polymorphism affects NO(x) concentrations in lead-exposed subjects. We studied 104 subjects exposed to lead who were non-smokers, 18-60 years of age, and not alcohol consumers. Genomic DNA was isolated from blood samples and genotypes for the T(-786)C polymorphism were determined by PCR and restriction fragment length digestion. Circulating NO(x) was determined by chemiluminescence. B-Pb and P-Pb were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. No significant correlations were found between NO(x) and B-Pb and P-Pb measured in the 104 subjects (all P > 0.05). However, while no significant correlation was found in subjects with TT genotype, a negative correlation was found between plasma NO(x) and B-Pb (r = 0.230, P = 0.048) and P-Pb (r = 0.194, P = 0.110) in subjects from TC + CC genotypes group. Our study shows a negative correlation between plasma NO(x) concentrations and B-Pb in carriers of the "C" allele, thus suggesting a possible mechanism possibly involved in lead exposure-induced increase in the susceptibility to cardiovascular diseases.     

Plasma and erythrocyte lipid peroxide levels in workers with occupational exposure to lead

The plasma and erythrocyte lipid peroxide levels were measured in a group of male subjects occupationally exposed to lead for an average period of 17 yr, and compared to those from an age-matched control group living in the same city in a similar socioeconomical environment. The blood lead and plasma zinc levels were measured by atomic absorption spectroscopy. The plasma and erythrocyte lipid peroxide levels were established by the malondialdehyde determination method. Significant differences were found in the blood lead levels in lead-exposed workers, 15.00±10.15 μg/dL as compared to controls, 2.37±0.89 μg/dL. The plasma (2.67±0.69 μM) and erythrocyte (27.53±6.28 nmol/g Hb) lipid peroxide levels in workers with occupational exposure to lead were significantly higher than controls, 1.23±0.61 μM and 14.35±2.08 nmol/g Hb, respectively. There were no significant differences of the zinc levels in both groups. The blood lead levels had a statistically significant positive correlation with age and with duration of exposure in both groups, but showed no relationship to the corresponding blood zinc levels. The results presented in this study indicate that the increase of plasma and erythrocyte lipid peroxide levels in workers exposed to lead may be related to the lead concentration, age and duration of exposure.     

Cytochemical localization of phosphatase activity in vascular bundles and contiguous tissues of the leaf ofZea mays L.

Summary The cytochemical localization of phosphatase activity has been carried out on small and intermediate vascular bundles and contiguous tissues of the leaf ofZea mays L. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP, and with ADP and -GP. Reaction product (lead deposits) was observed on the plasma membrane of all cell types. It was invariably heavier on the plasma membranes of the bundle-sheath cells, vascular-parenchyma cells, and the thin-walled sieve tubes and their associated companion cells than on those of the mesophyll cells. Within the bundles, the heaviest lead deposits frequently were found on the plasma membranes of the thin-walled sieve tubes and the least amount (often lacking) on those of the thick-walled sieve tubes. Formation of reaction product was suppressed by NaF, vanadate, and molybdate but not by PCMBS (p-chloromercuribenzene sulfonic acid). The results of the substrate-specificity and inhibitor-sensitivity studies indicate that a nonspecific acid phosphatase was probably responsible for the deposition of the reaction product and not the plasma membrane H⁺-ATPase. These results, in addition to an evaluation of the pertinent literature, lead us to conclude that H⁺-ATPase activity has yet to be demonstrated unequivocally in association with the plasma membrane of phloem cells with lead precipitation procedures. Nevertheless, the differences in amounts of reaction product generally associated with the plasma membranes of the thick- and thin-walled sieve tubes of the maize leaf indicate that the two types of sieve tube differ from one another physiologically.     

Effect of lead on Na+, K(+)-ATPase activity in Penaeus indicus postlarvae

In vivo effect of lead on Na, K(+)-ATPase was studied in plasma membrane/mitochondrial fraction of P. indicus post-larvae (PL), exposed to 30 days to a sublethal concentration (1.44 ppm) of lead. A significant (P < 0.05) decrease in the enzyme activity was observed for exposed PL with respect to their controls at different intervals except 24hr. Further the substrate (ATP) and ion (Na+ and K+)-dependent kinetics of Na+, K(+)-ATPase was studied with the plasma membrane/mitochondrial fractions of control and 30 days exposed PL. The apparent KM and V(max). values were calculated to determine the nature of inhibition. Both the control and exposed PL showed almost the same apparent KM values in the presence of different substrate or ion concentrations indicating that lead interacts with the enzyme at a different binding site.     

The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta.

The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na+, K+-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg2+-ATPase). Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor. It is concluded that the strontium capture technique gave a reliable localization for Na+, K+-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

Comparison of the toxicity and distribution of cadmium and lead in plant cells

Summary. The toxicity of heavy metals (Cd, Zn, and Pb) was assessed by in vivo observations of their effect on cytoplasmic streaming in Allium cepa L. bulb scale epidermal cells. On the basis of our results, the order of toxicity of the studied cations is Zn < Pb ≪ Cd. The difference in toxicity between cadmium and lead was found to be very large. When cytoplasmic streaming was assessed, this difference was threefold. When the total content of cadmium and lead (determined by inductively coupled plasma mass spectrometry) was the criterion, the difference in toxicity was 15-fold. Fractionation of the tissue and enzymatic digestion of the cells revealed that the largest proportion of cadmium was located in the cell walls (56%), whereas almost all of the lead (97.6%) was accumulated in an insoluble form. The speciation of water-soluble Pb and Cd fractions is discussed on the basis of analysis by capillary zone electrophoresis interfaced with inductively coupled plasma mass spectrometry of water extracts from epidermal cells. Lead and cadmium appeared to be bound mainly to salts, which explains their toxicity. Cadmium was complexed (detoxified) by organic acids, while thiols were the metal-complexing species for lead. Histidine formed complexes with both cadmium and lead. Ultrastructural analyses showed that lead was encapsulated in small vesicles in the cytoplasm. Fluorescence studies of the endoplasmic reticulum (ER) revealed that it underwent extensive fragmentation under the influence of lead, with numerous ER vesicles appearing in the cells. In other words, the lead deposits in the cytoplasm were contained in vesicles arising from fragmentation of the ER. These observations indicate that epidermal cells have a rapid and effective mechanism for detoxifying lead involving the ER, and this may be one of the mechanisms accounting for the lower toxicity of lead in comparison with cadmium. The suitability of Allium cepa bulb scale epidermal cells for use in ecotoxicological studies is also discussed. Step-by-step directions for this test are given. Correspondence and reprints: Department of Ecotoxicology, Institute of Experimental Plant Biology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.     

Cytochemical localization and quantification of plasma membrane Ca2+-ATPase activity in mollusc digestive gland cells

A cytochemical method allowing the localization and quantification of plasma membrane Ca2+-ATPase (PMCA) in frozen sections obtained from digestive gland cells of Mytilus galloprovincialis, Tapes tapes and Chamelea gallina, is presented. The method utilizes lead as a trapping agent of PO4(2-) ions released by Ca2+-ATPase activity. The amount of lead sulphide precipitate proportionally related to PMCA activity was quantified by a light microscopy digital imaging analysis system. The optimal assay conditions of Ca2+-ATPase activity evaluated at pH 7.4 were: 200 microM free Ca2+, 200 mM KCl, 2 mM ATP, and under such analysis conditions the enzyme showed a linear trend up to 60 min (at 20 degrees C). The PMCA activity was substrate specific: ADP was utilized only at a low rate (24% with respect to an equimolar ATP concentration), while glucose-6-phosphate and beta-glycerophosphate were poorly hydrolyzed. The enzyme activity was strongly inhibited by sodium ortho-vanadate. Our detection of a Ca2-ATPase activity at nanomolar concentrations of free Ca2+ suggests that we have identified a plasma membrane Ca2-ATPase involved in Ca2+ homeostasis. The Ca2+-ATPase was found to be localized in the basal part of the plasma membrane in the digestive gland cells of Mytilus galloprovincialis and Tapes tapes, but in the apical plasma membrane of Chamelea gallina. The possible implications of the different cellular distributions of PMCA activity is discussed.     

Effect of cold plasma on the E. coli cell wall and plasma membrane

The effect of cold plasma on E. coli cells was studied. It was shown that the treatment of E. coli cells with cold plasma caused partial or total disruption of the plasma membrane integrity, which was accompanied by a release of intracellular substances into the extracellular environment. A quantitative assessment of the extent of the damage to the cell membrane showed that a loss of no more than 23.6% of intracellular substances (calculated by the proportion of the intracellular nucleotide release) is sufficient to lead to cell death. The use of media with different ionic strength levels to create osmotic shock showed that the treatment of E. coli cells with cold plasma significantly decreased the cell wall strength.     

Effects of the administration of progesterone and adrenal medullectomy on the plasma fibrinogen levels in rats with surgical injury (laparotomy)

The probable r?le played by the adrenal medulla in the decrease of plasma fibrinogen due to the administration of progesterone (0.5 mg kg-1 day-1 during 72 h) in rats submitted to surgical injury (laparotomy) was studied. The results obtained lead to assume that the decrease of plasma fibrinogen observed in laparotomized rats injected with progesterone is indirectly produced through inhibition of the adrenal medulla. The action of progesterone on the plasma fibrinogen would be a pharmacological effect of the drug, since in doses of 0.10 mg kg-1 day-1 the decrease of the fibrinogen is not observed in laparotomized rats. The administration of progesterone in non injured rats does not modify the plasma fibrinogen as compared to the group of non injected rats.     

Lead concentrations in teeth from people living in Kosovo and Austria

The objective of this study was to compare lead concentrations in 86 human permanent teeth extracted from residents of three different geographical regions. The study included 31 permanent teeth from residents of Mitrovica (Kosovo), 32 from Klina (Kosovo) and 23 from Graz (Austria). The concentrations of lead were measured using Agilent 7500c inductively coupled plasma mass spectrometer (ICP-MS) (Agilent, Waldbronn, Germany). The comparisons between groups were based on the geographic area, age and gender. The highest lead level was found in teeth extracted from Mitrovica residents (22.3 mg/kg), followed by Klina (3.2 mg/kg), and Graz (1.7 mg/kg). Lead levels in teeth from Mitrovica residents are significantly higher (p < 0.0001) than in other two groups, possibly due to environmental contamination with lead. Overall results in this study support the concept that tooth lead level may present an important indicator in evaluating environmental exposure of human population to heavy metals.