Evaluation of three novel azasterols against Toxoplasma gondii.

Previous studies from our group have demonstrated the high susceptibility of Toxoplasma gondii tachyzoites to the sterol analogues 22,26-azasterol and 24,25-(R,S)-epiminolanosterol. In this work we present data on testing in vitro three novel azasterols as potential agents for the treatment of toxoplasmosis. The three compounds inhibited parasite growth at micromolar concentrations, in a dose-dependent manner. Electron microscopy analysis of intracellular tachyzoites after treatment with the most effective compound showed drastic mitochondrion swelling associated with the appearance of an electron-lucent matrix and disrupted cristae. Parasite lysis also took place. The appearance of electron dense cytoplasmic structures similar to amylopectin granules distributed throughout the parasite suggests that azasterols might be inducing differentiation of those tachyzoites which were not lysed to the bradyzoite stage.

Acetazolamide-based fungal chitinase inhibitors.

Chitin is an essential structural component of the fungal cell wall. Chitinases are thought to be important for fungal cell wall remodelling, and inhibition of these enzymes has been proposed as a potential strategy for development of novel anti-fungals. The fungal pathogen Aspergillus fumigatus possesses two distinct multi-gene chitinase families. Here we explore acetazolamide as a chemical scaffold for the inhibition of an A. fumigatus 'plant-type' chitinase. A co-crystal structure of AfChiA1 with acetazolamide was used to guide synthesis and screening of acetazolamide analogues that yielded SAR in agreement with these structural data. Although acetazolamide and its analogues are weak inhibitors of the enzyme, they have a high ligand efficiency and as such are interesting leads for future inhibitor development.

Novel functionalized melamine-based nitroheterocycles: synthesis and activity against trypanosomatid parasites.

Human African trypanosomiasis (HAT), caused by the protozoan parasite Trypanosoma brucei spp., is a major health problem in sub-Saharan Africa. New drugs are urgently required for the disease. Selective uptake of toxic compounds into trypanosomes has been achieved by exploiting plasma membrane transporters. For example, the P2 aminopurine transporter, along with other transporters, selectively concentrates melamine and benzamidine moieties into trypanosomes. We have previously reported the use of the melamine motif to selectively target nitrofuran to the trypanosome. In this paper we report the further investigation of the structure activity relationships and the effect of the introduction of different functionalized substituents onto the melamine unit. Most of the compounds tested in vitro for their trypanocidal activity showed activities in the submicromolar range against T. b. rhodesiense.

Quinuclidine derivatives as potential antiparasitics.

There is an urgent need for the development of new drugs for the treatment of tropical parasitic diseases such as Chagas' disease and leishmaniasis. One potential drug target in the organisms that cause these diseases is sterol biosynthesis. This paper describes the design and synthesis of quinuclidine derivatives as potential inhibitors of a key enzyme in sterol biosynthesis, squalene synthase (SQS). A number of compounds that were inhibitors of the recombinant Leishmania major SQS at submicromolar concentrations were discovered. Some of these compounds were also selective for the parasite enzyme rather than the homologous human enzyme. The compounds inhibited the growth of and sterol biosynthesis in Leishmania parasites. In addition, we identified other quinuclidine derivatives that inhibit the growth of Trypanosoma brucei (the causative organism of human African trypanosomiasis) and Plasmodium falciparum (a causative agent of malaria), but through an unknown mode(s) of action.

Evaluation of azasterols as anti-parasitics.

In this article, the design and synthesis of some novel azasterols is described, followed by their evaluation against Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani, and Plasmodium falciparum, the causative agents of human African trypanosomiasis, Chagas disease, leishmaniasis, and malaria, respectively. Some of the compounds showed anti-parasitic activity. In particular, a number of compounds appeared to very potently inhibit the growth of the blood stream form T. b. rhodesiense, with one compound giving an IC50 value of 12 nM. Clear structure activity relationships could be discerned. These compounds represent important leads for further optimization. Azasterols have previously been shown to inhibit sterol biosynthesis in T. cruzi and L. donovani by the inhibition of the enzyme sterol 24-methyltransferase. However, in this case, none of the compounds showed inhibition of the enzyme. Therefore, these compounds have an unknown mode of action.

New azasterols against Trypanosoma brucei: role of 24-sterol methyltransferase in inhibitor action.

A series of azasterol derivatives, designed as potential inhibitors of the Delta(24)-sterol methyltransferase enzyme (24-SMT), were synthesized and evaluated for their activities against parasitic protozoa. Values in the nanomolar range were obtained for 50% effective dose against the Trypanosoma brucei subsp. rhodesiense bloodstream form cultured in vitro. In order to investigate the mode of action, Trypanosoma brucei subsp. brucei 24-SMT was cloned and overexpressed and compounds were assayed for inhibitory activity. None of the inhibitors tested appeared to be active against the enzyme. Sterol composition analysis showed that only cholestane type sterols are present in membranes of bloodstream forms while ergosterol is a major component of procyclic sterol extracts. Interestingly, Northern blot analysis showed the presence of 24-SMT mRNA in both the procyclic and the bloodstream forms of the parasite, although levels of mRNA were threefold lower in the latter. Likewise, Western blot analysis and activity determinations evidenced the existence of active enzyme in both forms of the parasite. We conclude that the designed compounds act at sites other than 24-SMT in Trypanosoma brucei.

Preparation of transition-state analogues of sterol 24-methyl transferase as potential anti-parasitics.

There is an urgent need for new drugs to treat leishmaniasis and Chagas disease. One important drug target in these organisms is sterol biosynthesis. In these organisms the main endogenous sterols are ergosta- and stigmata-like compounds in contrast to the situation in mammals, which have cholesterol as the sole sterol. In this paper we discuss the design, synthesis and evaluation of potential transition state analogues of the enzyme Delta24(25)-methyltransferase (24-SMT). This enzyme is essential for the biosynthesis of ergosterol, but not required for the biosynthesis of cholesterol. A series of compounds were successfully synthesised in which mimics of the S-adenosyl methionine co-factor were attached to the sterol nucleus. Compounds were evaluated against recombinant Leishmania major 24-SMT and the parasites L. donovani and Trypanosoma cruzi in vitro, causative organisms of leishmaniasis and Chagas disease, respectively. Some of the compounds showed inhibition of the recombinant Leishmania major 24-SMT and induced growth inhibition of the parasites. Some compounds also showed anti-parasitic activity against L. donovani and T. cruzi, but no inhibition of the enzyme. In addition, some of the compounds had anti-proliferative activity against the bloodstream forms of Trypanosoma brucei rhodesiense, which causes African trypanosomiasis.