Interaction of phosphonomethyl analog of dihydroxyacetone phosphate with rabbit muscle aldolase.

Aldolase presents the same binding affinity for dihydroxyacetone phosphate and its phosphonomethyl analog, but the partition coefficient between the intermediates from the Michaelis complex to the eneamine is different. The effects of the structural modification of the triose phosphate substrate on the interaction with rabbit muscle aldolase are discussed in connection with the mechanistic pathway and the three-dimensional structure of the enzyme.

Inhibition of the glycolytic enzymes in the trypanosome: an approach in the development of new leads in the therapy of parasitic diseases.

Glycolysis in the trypanosome represents an important target for the development of new therapeutic agents due to the fact that this metabolism is essential for the parasite, glucose being its sole source of energy. In addition, different features of this metabolism and those associated with glycolytic enzymes offer opportunities for the development of efficient and selective compounds. Examples are given in this work of inhibitors directed to the enzymes aldolase and glyceraldehyde-phosphate-dehydrogenase and also of molecules acting specifically on the clusters of basic amino-acids present at the surfaces of the glycolytic enzymes in the parasite.

Synthesis and activity of inhibitors highly specific for the glycolytic enzymes from Trypanosoma brucei.

Most glycosomal enzymes of Trypanosoma brucei carry a relatively high number of positive charges. In at least 3 of the enzymes some of the charges unique to these enzymes are concentrated in 2 distinct areas on the enzymes' surface, about 4 nm apart [4] and these positively charged structural elements have been suggested to be the site of interaction with the trypanocidal drug Suramin. We have synthesized a series of symmetrical long chain molecules with negative charges or strong dipoles at each end. Several of these compounds inhibited the glycosomal enzymes more strongly than Suramin. They also exhibited a specificity for the trypanosome enzymes, when compared with homologous enzymes from other organisms. By varying the chain length of the active compounds, a 4-nm distance between the molecules' extremes proved optimal for inhibition. Tetra-substituted compounds were better than di-substituted. Modifications introduced at the two ends indicated that a planar orientation, with an amide bond linking a phenyl ring to the chain, is preferred. Inhibition kinetics for some of the enzymes indicated the existence of multi-site interactions with the inhibitors.

Characterization of a benzyl-phenoxy-ethanamine binding protein in Trypanosoma equiperdum and the possible relation between binding affinity and trypanocidal activity.

A new family of benzyl-phenoxy-ethanamine derivatives has been assayed for trypanocidal activity. Using tritiated morpholino-benzyl-phenoxy-ethanamine as a probe, it is shown that this ligand is able to bind specifically to a protein contained in extracts of Trypanosoma equiperdum. The binding is saturable and of high affinity (KD = 4 nM: Bmax = 200 fmol (mg protein)-1). The in vitro activities of the investigated compounds against this parasite correlate with their affinities to the putative binding site. Moreover, using an azido functionalized morpholino-benzyl-phenoxyethanamine as photoprobe a major M(r) = 40,000 protein was specifically revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. This molecular weight corresponds with the previously observed value determined for the antioestrogen binding site protein of rat liver which has been shown to specifically bind antioestrogens of the triphenylethylene family and phenoxyethanamine derivatives.

Interactions between trypanocidal drugs and membrane phospholipids. A surface pressure, surface potential and electrophoretic mobility study.

Amphiphilic diphenyl methane derivatives exhibiting both antiproliferative and trypanocidal effects were studied with respect to their interactions with phospholipids, in monolayers and bilayers. These compounds, namely (4-benzyl)-phenoxy-2 trimethylammonium ethane iodide (D1), (4-tertiobutyl)-phenoxy-2 morpholinium ethane chloride (D2), and (4-benzyl)-phenoxy-2 morpholinium ethane chloride (D3), were shown to interact with phosphatidylcholine (PC) and phosphatidylserine (PS) in monolayers, as monitored by surface pressure and surface potential measurements. The film expansion of monolayers, on 10 mM NaCl subphase at pH 7.1, was more pronounced in the presence of D2 and D3 in the subphase before spreading of the lipids than with the injection of the drugs underneath a preformed film. Apparent binding constants of 10(4) M-1 were determined for both drugs from monolayer experiments. With D2 in the presence of PS, results of monolayer compressions and electrophoretic mobility measurements indicate binding of the drug to the lipid molecules only when the molecular area was large. D3 was shown to interact with PS, both in monolayers and bilayers, with a drug-to-lipid binding constant of about 2 x 10(4)M-1, as evaluated from electrophoretic mobility measurements on PS liposomes. These results, which indicate binding of these drugs to phospholipids in the order D2 less than D3, correlate with the biological activity of the drugs, and may account for the discrepancy observed between the drug concentrations required for biological and binding activities.

Uptake of the nitroimidazole drug megazol by African trypanosomes.

Megazol, CL 64,855 (2-amino-5-[1-methyl-5-nitro-2-imidazolyl]-1,3, 4-thiazole) has been shown to be extremely effective in clearing experimental infections of African trypanosomes. An unusual amino-purine transporter termed P2, implicated in the transport of both the diamidine and melaminophenyl arsenical classes of drug in Trypanosoma brucei, recognised chemical groups on compounds which are also present on megazol. Megazol interacted with this carrier protein, as judged by its ability to inhibit P2 adenosine transport and to abrogate in vitro arsenical-induced lysis in a dose-dependent manner. However, parasites resistant to melaminophenyl arsenical and diamidine drugs due to lack of the P2 transporter showed no resistance to megazol. This is because passive diffusion represented the major route of entry. Initial rates of uptake were not saturable within the limit of megazol's solubility and did not conform to thermodynamic precepts compatible with carrier-mediated uptake. Adenosine and other P2 transporter substrates, even at high concentration, had little impact on megazol uptake. Uptake was biphasic, with a very rapid equilibration across the membrane followed by a slower accumulation over time. The equilibration phase represented a simple passive diffusion, with the subsequent uptake probably being due to metabolism of the drug.

Enzymatic reduction studies of nitroheterocycles.

The nitroimidazole derivative Megazol is a highly active compound used against several strains of Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanomiasis). With the aim of gaining an insight into the probable mode of action, the interaction of Megazol with different redox enzymes was studied in comparison to that of Nifurtimox and Metronidazole. The three nitroaromatic compounds are reduced by L-lactate cytochrome c-reductase, adrenodoxin reductase, and NADPH:cytochrome P-450 reductase (EC 1.6.2.4), the efficiencies of the enzymatic reductions being roughly related to the reduction potentials of these pseudo-substrates. As the enzyme responsible for the reduction of Megazol within the parasite has not yet been identified, the nitroimidazole was assayed with T. cruzi lipoamide dehydrogenase and trypanothione reductase. Megazol did not inhibit the physiological reactions but proved to be a weak substrate of both flavoenzymes. The single electron reduction of the compound by NADPH:cytochrome P-450 reductase, by rat liver as well as by trypanosome microsomes was confirmed by ESR experiments. As shown here, Megazol interferes with the oxygen metabolism of the parasite, but its extra activity when compared to Nifurtimox may be related to other features not yet identified.

Atypical neutrophil alkaline phosphatase associated with impaired neutrophil functions.

We report the case of a healthy young man presenting with atypical neutrophil alkaline phosphatase (NAP) and reduced neutrophil chemotactic activity, but with no susceptibility to infection. NAP activity was low, kinetic parameters were modified and immunoreactive properties and subcellular distribution were abnormal. Neutrophil morphology was normal. A similar pattern was observed in the patient's healthy brother. The profile of the observed anomalies offers some similarity to that previously described in patients with chronic myelogenous leukaemia. However, in the present case, the NAP deficiency with impaired neutrophil function was present in two brothers with no haematological symptoms and is probably related to a non-acquired neutrophil abnormality. This observation of a primary NAP variant reinforces the hypothesis of a direct link between NAP activity and functional properties of neutrophils.

Effect of megazol on Trypanosoma brucei brucei acute and subacute infections in Swiss mice.

Human African trypanosomiasis (HAT) or sleeping sickness is a major public health problem in 36 sub-Saharan African countries and is caused by Trypanosoma brucei gambiense and T. b. rhodesiense. About 25,000 new cases of the disease are reported annually, and around 50 million people are classed as at risk of contracting the disease. Until now; the only effective drug available for treatment of advanced HAT was the trypanocide melarsoprol. The mortality rate of melarsoprol treated patients is 1-5%. Megazol is a nitroimidazole derivative shown to be effective in vitro against T. b. brucei with an EC50 of 0.01 micrograms.ml-1. When this compound was tested for its in vivo activity in T. b. brucei infected Swiss mice, it was shown to cure the acute disease. However, megazol alone did not cause cure of mice carrying a subacute infection with involvement of the central nervous system (CNS). Combined suramin and megazol treatment did prove effective and the mice were shown to have remission without further relapse from the CNS. The study of three megazol derivatives is also described here. Substitution of a bromine, methyl or trifluoromethyl moiety at the 4 position of the imidazole ring abolished trypanocidal activity both in vivo and in vitro. Intermediates of megazol synthesis (imidazole sulfoxide and imidazole sulfone) were also tested, but were shown not to be active. It is thought that megazol trypanocidal effect may be due to the triggering of radical production by the compound, which have toxic effects on the trypanosomes metabolism. In depth study of megazol is needed to fully elucidate its pharmacokinetics and to precisely pin down its mode of action.

Further evidence for a biological role of anti-estrogen-binding sites in mediating the growth inhibitory action of diphenylmethane derivatives.

Several diphenylmethane derivatives have been synthesized with variable affinities for Anti-estrogen Binding Sites (ABS) but not for the estrogen receptor. Using these molecules as probes it is shown that their binding affinities for ABS correlate with their abilities to inhibit the growth of MCF-7 human breast cancer cells. In contrast they have no influence on the proliferation of tamoxifen-resistant variant cells (RTx6) in which ABS are undetectable. These data support the conclusion that ABS has a functional role in the anti-proliferative effect of triphenylethylene anti-estrogens and structurally related compounds.

Inhibition by sodium thiophosphate and L-p-bromotetramisole of neutrophil alkaline phosphatase in normal and trisomy 21 pregnancies.

Alkaline phosphatase concentrations are known to increase in blood neutrophils of normal pregnant women. The main kinetic parameters of this enzyme were analysed and compared in a group of 30 women with normal pregnancies and a group of 11 women whose fetuses had trisomy 21 (Down's syndrome = DS). The subjects were studied at an identical stage of gestation. Significant changes occurred in thermal stability and urea resistance in cases of DS pregnancies. We also investigated the inactivation constants for two chemicals: L-p-bromotetramisole, an uncompetitive inhibitor, and sodium thiophosphate, a competitive inhibitor. Ki measured for the two inhibitors were found to be significantly lower in cases of pathological pregnancies. The patterns observed in inhibition constants extend the biochemical characteristics of the atypical isoenzyme expressed in neutrophils of women with DS pregnancies.

An easy stereospecific synthesis of 1-amino-2,5-anhydro-1-deoxy-D-mannitol and arylamino derivatives.

1-Amino-2,5-anhydro-1-deoxy-D-mannitol and a series of arylamino derivatives were prepared by nitrous acid deamination of 2-amino-2-deoxy-D-glucose and subsequent reductive amination of the resulting 2,5-anhydro-D-mannose. Some of these compounds showed an enhanced affinity for the hexose transporter of Trypanosoma brucei as compared to D-fructose.

Uptake of NO-releasing drugs by the P2 nucleoside transporter in trypanosomes.

Nitric oxide (NO.) has been identified as a principal regulatory molecule of the immune system and the major cytotoxic mediator of activated immune cells. NO. can also react rapidly with a variety of biological species, particularly with the superoxide radical anion O2.- at almost diffusion-limited rates to form peroxynitrite anion (ONOO-). ONOO- and its proton-catalyzed decomposition products are capable of oxidizing a great diversity of biomolecules and can act as a source of toxic hydroxyl radicals. As a consequence, a strategy for the development of molecules with potential trypanocidal activities could be developed to increase the concentration of nitric oxide in the parasites through NO.-releasing compounds. In this way, the rate of formation of peroxynitrite from NO. and O2.- would be faster than the rate of dismutation of superoxide radicals by superoxide dismutases which constitute the primary antioxidant enzymatic defense system in trypanosomes. The adenosine transport systems of parasitic protozoa, which are also in certain cases implicated in the selective uptake of active drugs such as melarsoprol or pentamidine, could be exploited to specifically target these NO.-releasing compounds inside the parasites. In this work, we present the synthesis, characterization and biological evaluation of a series of molecules that contain both a group which would specifically target these drugs inside the parasites via the purine transporter, and an NO.-donor group that would exert a specific pharmacological effect by increasing NO level, and thus the peroxynitrite concentration inside the parasite.

[Rational concepts and the study of active molecules against various trypanosomiases]. / Conception rationnelle et étude de molécules actives contre les différentes trypanosomiases.

Several sets of compounds, active against different trypanosomes, Trypanosoma brucei and Trypanosoma cruzi are presented, the lethal doses for some of them being less than the micro-molar concentration. These compounds are designed by taking advantage of two metabolic features of these parasites, glucose metabolism and oxidative stress.

[An efficacy trial on Trypanosoma brucei brucei of molecules permeating the blood-brain barrier and of megazol]. / Essais d'efficacité sur Trypanosoma brucei brucei de molécules franchissant la barrière hématoméningée et du mégazol.

Human African trypanosomiasis (HAT) is a major public health problem in 36 sub-Saharan African countries and around 50 million people are classed as "at risk". About 25,000 new cases of the disease are reported annually by the World Health Organisation (WHO). This disease is fatal if untreated. As for now, chemotherapy is unsatisfactory and relies on a few drugs which show two major problems. The first is pharmacokinetics involving the passage through the blood-brain barrier. The second concerns toxicity and adverse side-effects of drugs used to treat this disease. New trypanocides should be safe, effective without toxicity. This study reports the action of 45 drugs, known to pass through the blood-brain barrier and belonging to different therapeutic classes, and also the megazol, a nitrothiadiazole derivative, on Trypanosoma brucei brucei AnTat 1-9 in vitro in acellular semi-defined medium. Results showed that some drugs did not modify the parasitic growth, and others were either trypanostatic or trypanocide. These last drugs were tested in vivo on T. b. brucei An-Tat 1-9 infected Swiss mice. Only megazol was shown to be effective and trypanocide. This compound might trigger the production of oxygen derivatives and free radicals-which have toxic effects on the trypanosome metabolism.

[Evaluation of an activity score of prehospital medicine: activity scoring using Smur (CAS)]. / Evaluation d'un score d'activité en médecine préhospitalière: codage d'activité Smur (CAS).

OBJECTIVE: The activity of prehospital emergency medicine (PEM) teams is mainly assessed by the amount of medical interventions and their duration. However, these two parameters do not reflect workload correctly. The aim of this study was to compare a new activity scoring system for PEM teams (CAS) with the standard TISS score. STUDY DESIGN: Prospective comparative study. PATIENTS: This study included 4,650 patients, with a median age of 39 years [0-100], attended by PEM teams during 4,189 ambulance transports (83% primary transports and 17% interhospital transfers). METHODS: The CAS score derived from Omega scoring system is the sum of 51 items specifically suited for PEM, each one being rated 1, 3, 6 or 10. CAS and TISS, were prospectively determined for all medical ambulance transports over 1.5 year. Transport data and main diagnosis were collected. Results were analysed with non parametric statistical tests. RESULTS: Median duration of interventions (39 min [0-475]) and median CAS score (7 [0-72]) were comparable (R' = 0.38, P < 0.001). Median TISS was 3 [0-30]. CAS score was correlated with TISS (R' = 0.92, P < 0.001). CAS score was higher in men than in women (7 [0-72] vs 7 [0-45], P < 0.001). CAS scores in patients with cardiologic (11 [0-72]), respiratory (9 [0-31]) or neurological insults (9 [0-43]) were significantly higher than those of patients with other insults (P < 0.001). CONCLUSION: The CAS scoring system is a valuable indicator of medical team prehospital workload, probably more suited for prehospital emergency medicine than TISS.

Design of a Physical Activity Program to Prevent Functional Decline in Onco-Geriatric Patients (CAPADOGE): A Randomized Multicenter Trial.

BACKGROUND: Cancer in older patient favours the development of frailty: feeling of exhaustion, loss of weight, decreased muscle strength, slow gait speed, and low physical activity. OBJECTIVES: To evaluate the efficacy of adapted physical activity phone advices in limiting the cancer-induced loss of autonomy and frailty phenotype development. DESIGN: Multicenter randomized controlled trial. SETTING: Patients (>70y) undergoing curative treatment for cancer (n=400) will be recruited from 12 centres. INTERVENTION: The intervention consists in phoned personalized physical activity advices related to strength, aerobic, balance, proprioception, and flexibility. The contacts are performed twice a month during six months and then monthly until 1 year. The intervention complements the PNNS booklet advices (National Nutritional Health Program). The trial compares «individualized phone advices + PNNS¼ to «usual care + PNNS¼. MEASUREMENTS: Functional, cognitive, clinical and self-reported data are assessed before treatment and at 3, 6, 12, 18, and 24 month follow-up. The primary outcome is the proportion of subjects with a one-year decreased SPPB (Short Physical Performance Battery) score of one point or more, as compared to baseline. The secondary outcomes include quality of life items, rate of hospitalizations, institutionalizations, mortality, Fried phenotype at 1 and 2 years, and the SPPB score at 2 years. DISCUSSION: This large trial will provide clinical data of the effects of an exercise advices intervention in older patients during cancer therapy on function and cognition evolution, and quality of life. The possibilities of minimizing the development of frailty phenotype due to these advices will be explored.