New Azasterols against Trypanosoma brucei : Role of 24-Sterol Methyltransferase in Inhibitor Action

Abstract

A series of azasterol derivatives, designed as potential inhibitors of the ⌬ 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.

Quinuclidine Derivatives as Potential Antiparasitics

“…The bloodstream form of T. brucei is thought to scavenge sterols from the human host, although procyclic T. brucei has an active ergosterol biosynthetic pathway that includes the enzyme SQS. Interestingly, we have recently shown that although the bloodstream form of T. brucei scavenges sterols from the human host and does not synthesize ergosterol, it does expresses sterol 24-methyltransferase, another enzyme involved in ergosterol biosynthesis (14). P. falciparum, in contrast, lacks the enzymes involved in sterol biosynthesis beyond farnesyl diphosphate synthase, as inferred from genomic data.…”

SAR studies on azasterols as potential anti-trypanosomal and anti-leishmanial agents

“…[18][19][20] We have also prepared some squalamine analogues which have some anti-leishmanial activity. 21 These azasterols that we have reported [10][11][12][13][14] have interesting in vitro activity against T. brucei rhodesiense and Leishmania donovani. From the evaluation of these compounds it was possible to construct a preliminary pharmacophore for the activity against T. brucei rhodesiense.…”

“…1). [10][11][12][13][14] We noticed that some of these compounds were active against bloodstream form (bsf) T. brucei; however bsf T. brucei is reported to scavange cholesterol from the host, rather than biosynthesise ergosterol, 15,16 suggesting alternate modes of action for these compounds. We also discovered that some compounds had multiple modes of action against Leishmania spp.…”

“…Furthermore, we demonstrated that whilst some azasterols inhibited the growth of T. brucei with an EC 50 of less than 50 nM, they did not inhibit 24-SMT, suggesting an alternative mode of action. 14 Interestingly other sterol-amine conjugates have been shown to have antimicrobial activity. A number of sterols with basic side chains have been shown to have antimicrobial activity; 17 this includes a natural product, squalamine, that has been shown to have potent antimicrobial activity.…”

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack

“…Human farnesyl diphosphate synthase is the target of bisphosphonates (e.g., tiludronate), used against osteoporosis ( 10 ). Squalene epoxidase and sterol 24-methyltransferase are antifungal targets, inhibited by allylamines (e.g., terbinafi ne) and azasterols, respectively (11)(12)(13)(14). A particularly promising target is sterol 14-demethylase (CYP51).…”