Anti-parasitic drug discovery takes a giant leap forward

Although rare, parasitic infections can be severe and cause death. Presently, there is a paucity of compounds to treat these infections. identified two suicide substrate inhibitors directly inhibiting the methyltransferase Acanthamoeba that these steroids 1) covalently bound and inhibited sterol C28-methyltransferase highly inhibitory to trophozoite growth (IC and were to mammalian cells.

Acanthameoba spp.have a rudimentary life cycle consisting of trophozoite and cyst stages (Fig. 1).The trophozoite phase predominates when nutrients are plentiful, while nutrient depletion or drug treatment drives the initiation of the cyst phase (2).Trophozoites infect human hosts and are disease spreading, while the cyst phase is dormant and protects the organism against host responses.Anti-Acanthameoba treatments are severely limited and include milfetosine, biguanide, and voriconazole (3).
Both Acanthameoba SMTs are more orthologous to plant SMT enzymes, rather then those of trypanosomes.This sets up the unique opportunity for species-specific antiameobic treatment.
The authors started off their journey by asking the important question of whether CHT and ERGT were worth pursuing as steroidal inhibitors.
They tested if these steroids inhibited trophozoite growth in cell culture.Compounds were tested against Acanthameoba castellanii trophozoites.Importantly, they found that CHT and ERGT were highly potent inhibitors with IC 50 and MAC values of 51nM and 5µM, respectively.Wash out experiments, whereby each steroid was removed after a period of time and growth was then monitored, showed that trophozoites were still unable to grow even in the absence of either steroid, giving the authors the first hint that CHT and/or ERGT may act as suicide substrate inhibitors.Finally, they found that neither steroid was cytotoxic to HEK293 mammalian cells.
Based on these very positive results, they next performed elegant in vitro enzymatic assays, linked to extensive GC/MS product identification, to both characterize the CHT-and ERGT-derived products formed, and gain insight into their mechanisms of action.Ac24-SMT was able to convert CHT to the single product, ERGT, whereas ERGT itself did not productively bind to the enzyme.On the other hand, Ac28-SMT converted CHT to multiple sterol species, including C 28 and C29 sterols, C28 and C29 monols, and C28-C29-steroidal diols.Overall, the types of products formed supported the hypothesis that they act as suicide substrates, forming an irreversible covalent complex with Ac28-SMT.The authors postulated that the covalent interaction was stabilized by the conjugated double bond in each analog.
Next, the authors used site-directed mutagenesis and converted the conserved Tyr60 and Tyr64 residues found within region 1 of Ac24-SMT and Ac28-SMT, respectively, to explore mechanistically their importance in appropriate substrate binding and product formation.Each Tyr was converted to a Phe or Leu, and products were identified using CTO (cholesta-5,7,24-trienol), CHT and ERGT as substrates.CTO is used as an in vitro and in vivo and served as a control substrate for product formation.
Interestingly, mutation of Ac24-SMT Tyr60 to either Phe or Leu did not alter the product species formed using CTO or CHT as substrates, when compared those produced by wild type Ac24-SMT.On the other hand, mutating Tyr64 to Phe within Ac28-SMT caused dramatic shifts in the ratios of products formed using CTO as a substrate.The data supported the idea that Tyr64 was essential for the sequential first and second C 1 transfer reaction.Products formed when CHT or ERGT were used as substrates showed a severe reduction in the products that would be seen if Ac28-SMT was active.The substitution of Leu total abolished activity, as ERGT was not converted to any product(s).
Finally, they obtained more direct evidence that CHT and ERGT were irreversible inhibitors, evidenced by the fact that when CHT or ERGT were used as substrates for Ac28-SMT, the products formed were species that would be seen only if the methylation reactions were inhibited.Km and kcat values obtained for CHT were similar to those of the natural substrate, methylenephenol.They also showed that CHT had IC50 and ki values for Ac28-SMT that were similar to those seen for related SMTs.Finally, they went on to show that high concentrations of methylenephenol protected Ac28-SMT from inactivation, further validating the hypothesis of an irreversible mechanism of action occurring at the active site.
The Nes laboratory has had a long-standing expertise in identifying and biochemically characterizing sterol biosynthesis inhibitors targeted against many pathogenic microorganisms (4-7).In fact, they recently characterized steroidal transition state and suicide substrate inhibitors targeting SMTs (24(R,S),25-epiminolanosterol (EL) and 26,27dehydrolanosterol (DHL), respectively) (8).These past studies have laid the foundation for the present work described in Zhou et al., (1), whereby two novel suicide substrate inhibitors have been studied as potential efficacious antiparasitic therapeutics.
Based on work presented by Zhou et al., (1) these steroids can now be added to the ever-growing inhibitor catalog of the Nes laboratory.The important work in Zhou et al., (1) will undoubtedly increase the optimism of these steroidal inhibitors becoming bona fide antiparasitic therapeutics in the near future.Results showing their efficacy in murine infection models would seal the deal.