Design and synthesis of novel spirooxindole-indenoquinoxaline derivatives as novel tryptophanyl-tRNA synthetase inhibitors.

In the current study, we used an integrated approach combining bioinformatics, rational drug design, one-pot synthesis, and biological experiments in vitro for the potential discovery of novel tryptophanyl-tRNA synthetase (TrpRS) inhibitors. Atom economic and diastereoselective syntheses were used to generate several Spirooxindole-indenoquinoxaline derivatives in situ from isatin and amino acids viz. proline, phenylglycine, and sarcosine through targeting the 1,3-dipolar cycloaddition of azomethine ylides. These compounds were assayed by biochemical TrpRS inhibition, using in vitro experiments to test against various gram-positive and gram-negative strains, and using diffuse large B cell lymphoma (DLBCL) cell lines. Compound 6e was found to be the most active in vitro with IC50 values of 225 and 74 nM for tests against hmTrpRS and ecTrpRS, respectively. We also found a MIC90 value of 4 µg/mL for tests against S. aureus and IC50 values which ranged from 2.9 to 4.8 µM for tests against proliferation of DLBCL cell lines. Moreover, compound 6e was remarkably good at inducing bacterial autolysis in MRSA strains. Our results suggested that such an integrated approach could be an attractive and viable strategy for the discovery of novel TrpRS inhibitors as potential lead compounds for antibiotics and as novel anticancer agents. Discovery of novel spirooxindole-indenoquinoxaline TrpRS inhibitors as potential lead compounds with antibacterial and antitumor activities.

Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies.

Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation.

Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment.

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that ß2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21 days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4 mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9 days of treatment, while hypertrophy was observed only in EDL after 9 days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14 days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.

[Rhabdomyolysis and paraphenylene-diamine poisoning]. / Rhabdomyolyse et intoxication à la paraphénylène-diamine.

OBJECTIVE: The aim of this study is to describe clinical description, biological findings, outcome and prognostic factors of paraphenylene-diamine poisoning. PATIENTS AND METHODS: We report a cohort study spreaded over 6 years (1999-2004), realized in Medical Intensive Care Unit in Ibn-Rochd University Hospital at Casablanca (Morocco). This study included 315 patients admitted for paraphenylene-diamine (PPD) poisoning. Diagnosis was based on: poisoning reported by the patient or his family, clinical data, biological findings and qualitative determination of PPD. Epidemiological parameters was obtained at admission. Every day, clinical and biological data, therapy and gravity scores were collected and a mean was calculated. RESULTS: 315 patients were admitted over this period. The mean age was 23+/-9 years. We noticed a clear female predominance (sex-ratio=9.86). The intoxication was voluntarily aiming at autolysis in 93.3% of the cases. The patients were admitted at about 5+/-5.3 hours after the intoxication. The clinical chart was at first dominated by the respiratory and renal symptoms. The mean of CPK was 132,351.8+/-164,978 UI/l. The treatment was especially symptomatic. The mortality was 47%. The multivariate analysis concluded that acid urinary pH, hyperglycaemia, hard muscles, betamimetic drugs and MPM II>0.14 were associated with a poor prognosis. CONCLUSION: The PPD poisoning represents the first cause of toxic rhabdomyolysis in our context and responsible of high mortality. For that, it's necessary to control PPD trade, to inform the medical persons and a rapid management.

Reduction in myocardial infarct size: prevention of heart cell death.

The course of myocardial necrosis, the clinical syndrome, and methods of treatment are presented. Heart cell death may be prevented by maintaining the balance between myocardial oxygen and energy supply and consumption. New technics of improving this balance by reducing myocardial energy demand, altering metabolism, increasing myocardial substrate supply, and protecting cellular integrity are discussed.

On the physiological functions of teichoic acids.

The choline-containing teichoic acids of pneumococci can be modified by biosynthetic replacement of the choline residues with certain structural analogues, such as ethanolamine (EA) or the N-monomethyl-(MEA) and N-dimethyl-(DEA) amino derivatives of ethanolamine. Cells containing such analogues in their teichoic acids develop pleiomorphic alterations in several physiological properties, which include resistance to detergent-induced lysis and inhibition of cell separation (chain formation). We report here the results of physiological studies on the mechanism of these two phenomena. Our results are summarized in the following: (a) Pneumococci grown on various amino alcohols produce cell walls of identical amino sugar and amino acid composition. (b) Both choline- and EA-containing teichoic acids seem to follow the same conservative pattern of segregation during growth and cell division.(c)Lysis sensitivity of pneumococci requires the juxtaposition oflysissensitive (choline-containing) cell walls and endogenous autolysin at the cell wall growth zone. (d) Upon readdition of choline to ethanolamine-containing cells, lysis sensitivity and catalytically active (C-type) autolysin reappear in the bacteria with the same kinetics. (e) The chains of EA-grown pneumococci contain fully compartmentalized cells and normal cross walls.

Influence of macromolecular biosynthesis on cellular autolysis in Streptococcus faecalis.

The addition of several different antibiotics to growing cultures of Streptococcus faecalis, ATCC 9790, was found to inhibit autolysis of cells in sodium phosphate buffer. When added to exponential-phase cultures, mitomycin C (0.4 mug/ml) or phenethyl alcohol (3 mg/ml) inhibited deoxyribonucleic acid synthesis, but did not appreciably affect the rate of cellular autolysis. Addition of chloramphenicol (10 mug/ml), tetracycline (0.5 mug/ml), puromycin (25 mug/ml), or 5-azacytidine (5 mug/ml) to exponential-phase cultures inhibited protein synthesis and profoundly decreased the rate of cellular autolysis. Actinomycin D (0.075 mug/ml) and rifampin (0.01 mug/ml), both inhibitors of ribonucleic acid (RNA) synthesis, also reduced the rate of cellular autolysis. However, the inhibitory effect of actinomycin D and rifampin on cellular autolysis was more closely correlated with their concomitant secondary inhibition of protein synthesis than with the more severe inhibition of RNA synthesis. The dose-dependent inhibition of protein synthesis by 5-azacytidine was quickly diluted out of a growing culture. Reversal of inhibition was accompanied by a disproportionately rapid increase in the ability of cells to autolyze. Thus, inhibition of the ability of cells to autolyze can be most closely related to inhibition of protein synthesis. Furthermore, the rapidity of the response of cellular autolysis to inhibitors of protein synthesis suggests that regulation is exerted at the level of autolytic enzyme activity and not enzyme synthesis.