Jacques Périé

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.

Cyclic voltammetric studies on nitro radical anion formation from megazol and some related nitroimidazole derivatives

Abstract Megazol (2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazol, CAS 19622-55-0) and related nitroimidazole compounds are being tested as antichagasic drugs. Little is known on the mode of action of megazol. However, there is evidence that one-electron reduction of megazol to the corresponding nitro radical anion is a key step in the reaction mechanism. Consequently, this paper is focused on the cyclic voltammetric behaviour of megazol and related nitroimidazole derivatives with the aim of revealing the formation and stability of the corresponding nitro radical anions. All the compounds studied produce a well resolved nitro/nitro radical anion couple. The resolution of the couple was improved with the addition of tetrabutylammonium ions which hinders the protonation of the nitro radical anion at the electrode surface, thus enhancing the stability of the nitro radical anion. Only megazol produced a cyclic voltammogram distorted by the presence of a pre-peak due to strong adsorption of the corresponding nitro radical anion. The pre-peak occurs at potentials more positive than the diffusion controlled peak because the Gibbs energy of adsorption of the nitro radical anion makes the reduction of megazol to the adsorbed nitro radical anion easier than to the radical anion in solution. The sulphur atom in the thiadiazole ring plays a crucial role in the adsorption phenomena. Using the cyclic voltammetry theory for the disproportionation reaction, we have calculated the second-order decay rate constant, k2, and the half-life time, t1/2, for all the nitro radical anions of the studied nitroimidazole derivatives. The values obtained were compared with those of the corresponding nitro radical anions obtained from nifurtimox and benznidazole, the classic antichagasic drugs. Also, our results show that cyclic voltammetry is a good alternative to the classic pulse radiolysis method to obtain reliable values of the E17 parameter for nitro radical anions.

Glycolysis and proteases as targets for the design of new anti-trypanosome drugs.

Glycolysis is considered as a promising target for new drugs against parasitic trypanosomatid protozoa, because this pathway plays an essential role in their ATP supply. Trypanosomatid glycolysis is unique in that it is compartmentalised, and many of its enzymes display specific structural and kinetic features. Structure- and catalytic mechanism-based approaches are applied to design compounds that inhibit the glycolytic enzymes of the parasites without affecting the corresponding proteins of the human host. For some trypanosomatid enzymes, potent and selective inhibitors have already been developed that affect only the growth of cultured trypanosomatids, and not mammalian cells. Examples are developed concerning all enzymes in the hexoses part with also others concerning glyceraldehyde-phosphate dehydrogenase and pyruvate-kinase for the trioses part. Concerning cysteine protease inhibitor development, a great number of irreversible alkylating agents have shown their efficacy towards the active site cysteine of parasite proteases. This includes fluoromethylketones, epoxides, diazomethylketones, vinylsulfones to mention a few. These functional groups are activated electrophiles that react with the nucleophilic cysteine of the active site and are generally quite selective for cysteine versus serine. They are thought to be also reactive to numerous other nucleophiles in the body, especially other thiols. This potentially hampering property seems not to be detrimental for two reasons: first a recent report has shown that cysteine protease inhibitors containing a vinylsulfone electrophile are unreactive towards thiols such as glutathione and can be considered to be inert in the absence of catalytic machinery. Secondly, irreversible inhibitors are shown to be less toxic than presumed in the parasite treatment, owing to some bioselectivity displayed by the parasite itself.

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.

Sequencing, Modeling, and Selective Inhibition of Trypanosoma brucei Hexokinase

For Trypanosoma brucei, a parasite responsible for African sleeping sickness, carbohydrate metabolism is the only source of ATP, and glycolytic enzymes are localized within membrane-bound organelles called glycosomes. Hexokinase, the first enzyme of the glycolytic pathway, was chosen as a target for selective drug design. We have cloned and sequenced the hexokinase gene of T. brucei. In parallel, we have synthesized several inhibitors. Kinetic analysis revealed differences in the binding mode of these compounds toward yeast and T. brucei hexokinases, while the m-bromophenyl glucosamide was found to be selective for T. brucei. The modeled structure of T. brucei hexokinase-inhibitor complex (using the crystal structure of the Schistosoma mansoni hexokinase as a template) allows us to propose a mode of action of this inhibitor for the trypanosome hexokinase and to account for the observed selectivity.

An improved chemical and enzymatic synthesis of new fructose derivatives for import studies by the glucose transporter in parasites

Abstract This paper presents the chemoenzymatic synthesis of D-fructose analogues substituted at position C6. These compounds are the unique products of rabbit muscle aldolase catalyzed aldolisation of D-glyceraldehyde analogues (obtained by stereospecific chemical synthesis) with DHAP, followed by a dephosphorylation step with acid phosphatase.

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 megazols 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.

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.

Synthesis of phosphono analogues of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate

The present paper describes the synthetic routes of six phosphono analogues of dihydroxyacetone phosphate and five phosphono analogues of glyceraldehyde 3-phosphate through alpha-, beta- and gamma-hydroxyphosphonate esters precursors containing a protected carbonyl group. In some situations, depending on the sequence used for the deprotection of the phosphonate and carbonyl groups, the aldol/ketol rearrangement allowed the synthesis of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate analogues from the same precursors. All these analogues are of interest both as active-site probes and as potential substrates for glycolytic enzymes such as fructose 1,6-diphosphate aldolases (EC 4.1.2.13).

Peroxynitrite-induced nitration of tyrosine-34 does not inhibit Escherichia coli iron superoxide dismutase.

The peroxynitrite anion is a potent oxidizing agent, formed by the diffusion-limited combination of nitric oxide and superoxide, and its production under physiological conditions is associated with the pathologies of a number of inflammatory and neurodegenerative diseases. Nitration of Escherichia coli iron superoxide dismutase (Fe-SOD) by peroxynitrite was investigated, and demonstrated by spectral changes and electrospray mass spectroscopic analysis. HPLC and mass studies of the tryptic digests of the mono-nitrated Fe-SOD indicated that tyrosine-34 was the residue most susceptible to nitration by peroxynitrite. Exclusive nitration of this residue occurred when Fe-SOD was exposed to a cumulative dose of 0.4 mM peroxynitrite. Unlike with human Mn-SOD, this single modification did not inactivate E. coli Fe-SOD at pH 7.4. When Fe-SOD was exposed to higher concentrations of peroxynitrite (7 mM), eight tyrosine residues per subunit of the protein, of the nine available, were nitrated without loss of catalytic activity of the enzyme. The pK(a) of nitrated tyrosine-34 was determined to be 7.95+/-0.15, indicating that the peroxynitrite-modified enzyme appreciably maintains its protonation state under physiological conditions.