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Anti-parasitic drug discovery takes a giant leap forward1

  • Joseph T. Nickels Jr.
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Institute of Metabolic Disorders, Genesis Biotechnology Group, Hamilton, NJ 08691, and Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, NJ 08901
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  • Author Footnotes
    1 See referenced article, J. Lipid Res. 2019, 60: 981–994.
Open AccessPublished:March 27, 2019DOI:https://doi.org/10.1194/jlr.C094250
      Although rare, parasitic infections can be severe and cause death. Presently, there is a paucity of compounds to treat these infections. Zhou et al. (
      • Zhou W.
      • Ramos E.
      • Zhu X.
      • Fisher P.M.
      • Kidane M.E.
      • Vanderloop B.H.
      • Thomas C.D.
      • Yan J.
      • Singha U.
      • Chaudhuri M.
      Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.
      ) have identified two steroidal suicide substrate inhibitors [cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT)] directly inhibiting the sterol methyltransferase activities of Acanthamoeba castellanii (AcSMTs), the organism causing blinding keratitis (BK) and granulomatous amebic encephalitis (GAE). They demonstrated that these steroids 1) covalently bound and inhibited sterol C28-methyltransferase (Ac28-SMT), 2) were highly growth inhibitory to trophozoite growth (IC50~nM), and 3) were nontoxic to mammalian cells. The future validation of these structures as bone fide anti-parasitic therapeutics will spark great interest in the future targeting of sterol biosynthesis for treating parasitic infections.
      Acanthamoeba spp. are ubiquitous free-living protozoa causing diseases of the eye, skin, and central nervous system (
      • Kot K.
      • Lanocha-Arendarczyk N.A.
      • Kosik-Bogacka D.I.
      Amoebas from the genus Acanthamoeba and their pathogenic properties.
      ). Acanthamoeba castellanii causes BK and GAE. Individuals with GAE are immune-compromised and although infections are rare, a high mortality rate is seen. The fact that Acanthamoeba spp. infections are on the rise clearly reinforces the need for drug discovery aimed at improving the production of novel and highly potent anti-parasitic therapeutics.
      Acanthamoeba 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 (
      • Kot K.
      • Lanocha-Arendarczyk N.A.
      • Kosik-Bogacka D.I.
      Amoebas from the genus Acanthamoeba and their pathogenic properties.
      ). Trophozoites infect human hosts and are disease spreading, while the cyst phase is dormant and protects the organism against host responses. Anti-Acanthamoeba treatments are severely limited and include milfetosine, biguanide, and voriconazole (
      • Siddiqui R.
      • Aqeel Y.
      • Khan N.A.
      The development of drugs against Acanthamoeba infections.
      ).
      Figure thumbnail gr1
      Fig. 1.Acanthamoeba castellanii life cycle and sterol biosynthetic pathway. Acanthamoeba castellanii can exist as a trophozoite when nutrients are plentiful or upon nutrient depletion, a dormant cyst. Trophozoites are the active disease-spreading stage. Trophozoite sterol synthesis proceeds by way of the conversion of the precursor cycloartenol to the end products ergosterol or 7-dehydroporiferasterol. Cycloartenol is converted to 24-methylene cycloartenol by the Ac24-SMT. Ac28-SMT converts methylene lophenol to 24(28)E-ethylidene lophenol. Methylene lophenol is a branch-point sterol used for the production of terminal sterols. Both SMT reactions are inhibited by CHT (
      • Zhou W.
      • Ramos E.
      • Zhu X.
      • Fisher P.M.
      • Kidane M.E.
      • Vanderloop B.H.
      • Thomas C.D.
      • Yan J.
      • Singha U.
      • Chaudhuri M.
      Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.
      ) and ERGT (
      • Kot K.
      • Lanocha-Arendarczyk N.A.
      • Kosik-Bogacka D.I.
      Amoebas from the genus Acanthamoeba and their pathogenic properties.
      ). Figure adapted from (

      Kidane, M. E., 2017. 26,27-dehydrolanosterol and cholesta-5,7,22,24-tetraenol are suicide substrates of sterol methyl transferase in Acanthamoeba castellanii. PhD Thesis. Texas Tech University, Lubbock, TX.

      ).
      The Acanthamoeba spp. sterol biosynthetic pathway uses cycloartenol as the precursor for C28-ergosterol and C29-7-dehydroporiferasterol end-product synthesis, rather then lanosterol that is used by Trypanosoma brucei (Fig. 1). The sterol 24- and C28-methyltransferases (Ac24-SMT and Ac28-SMT) are intricately involved in terminal synthesis. Ac24-SMT uses cycloartenol to synthesize 24-methylene cycloartenol, the precursor for the synthesis of the Ac28-SMT substrate 24(28)-methylene lophenol. This intermediate sits at a branch point, as it can be used as a precursor for ergosterol synthesis or shunted toward C29-7-dehydroporiferasterol formation. Both Acanthamoeba SMTs are more orthologous to plant SMT enzymes rather than those of trypanosomes. This sets up the unique opportunity for species-specific anti-ameobic treatment.
      Using a number of elegant kinetic and in vivo cell culture studies in association with inhibitor-product analysis, Zhou et al. (
      • Zhou W.
      • Ramos E.
      • Zhu X.
      • Fisher P.M.
      • Kidane M.E.
      • Vanderloop B.H.
      • Thomas C.D.
      • Yan J.
      • Singha U.
      • Chaudhuri M.
      Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.
      ) extensively interrogated the steroids cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT) as to their potential ability to target and inhibit Ac24-SMT and Ac28-SMT (Fig. 1). In total, this work has definitively established CHT and ERGT as steroidal suicide substrates of Ac28-SMT that can inhibit trophozoite cell growth with high potency.
      The authors started off their journey by asking the important question of whether CHT and ERGT were worth pursuing as steroidal inhibitors. They tested whether these steroids inhibited trophozoite growth in cell culture. Compounds were tested against Acanthamoeba castellanii trophozoites. Importantly, they found that CHT and ERGT were highly potent inhibitors with IC50 and minimal amoebicidal concentration values of 51 nM and 5 μM, respectively. Washout 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 C28 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 substrate 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 to 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 C1 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 totally abolished activity, as ERGT was not converted to any product(s).
      Finally, they obtained more direct evidence that CHT and ERGT were irreversible inhibitors, shown 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 longstanding expertise in identifying and biochemically characterizing sterol biosynthesis inhibitors targeted against many pathogenic microorganisms (
      • Haubrich B.A.
      • Singha U.K.
      • Miller M.B.
      • Nes C.R.
      • Anyatonwu H.
      • Lecordier L.
      • Patkar P.
      • Leaver D.J.
      • Villalta F.
      • Vanhollebeke B.
      Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth.
      ,
      • Zhou W.
      • Lepesheva G.I.
      • Waterman M.R.
      • Nes W.D.
      Mechanistic analysis of a multiple product sterol methyltransferase implicated in ergosterol biosynthesis in Trypanosoma brucei.
      ,
      • Leaver D.J.
      • Patkar P.
      • Singha U.K.
      • Miller M.B.
      • Haubrich B.A.
      • Chaudhuri M.
      • Nes W.D.
      Fluorinated sterols are suicide inhibitors of ergosterol biosynthesis and growth in Trypanosoma brucei.
      ,
      • Miller M.B.
      • Patkar P.
      • Singha U.K.
      • Chaudhuri M.
      • David Nes W.
      24-Methylenecyclopropane steroidal inhibitors: a Trojan horse in ergosterol biosynthesis that prevents growth of Trypanosoma brucei.
      ). In fact, they recently characterized steroidal transition state and suicide substrate inhibitors targeting SMTs (24(R,S),25-epiminolanosterol and 26,27-dehydrolanosterol, respectively) (
      • Kidane M.E.
      • Vanderloop B.H.
      • Zhou W.
      • Thomas C.D.
      • Ramos E.
      • Singha U.
      • Chaudhuri M.
      • Nes W.D.
      Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design.
      ). These past studies have laid the foundation for the present work described in Zhou et al. (
      • Zhou W.
      • Ramos E.
      • Zhu X.
      • Fisher P.M.
      • Kidane M.E.
      • Vanderloop B.H.
      • Thomas C.D.
      • Yan J.
      • Singha U.
      • Chaudhuri M.
      Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.
      ), whereby two novel suicide substrate inhibitors have been studied as potential efficacious anti-parasitic therapeutics.
      Based on work presented by Zhou et al. (
      • Zhou W.
      • Ramos E.
      • Zhu X.
      • Fisher P.M.
      • Kidane M.E.
      • Vanderloop B.H.
      • Thomas C.D.
      • Yan J.
      • Singha U.
      • Chaudhuri M.
      Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.
      ), these steroids can now be added to the ever-growing inhibitor catalog of the Nes laboratory. The important work in Zhou et al. (
      • Zhou W.
      • Ramos E.
      • Zhu X.
      • Fisher P.M.
      • Kidane M.E.
      • Vanderloop B.H.
      • Thomas C.D.
      • Yan J.
      • Singha U.
      • Chaudhuri M.
      Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.
      ) will undoubtedly increase the optimism that these steroidal inhibitors will become bona fide anti-parasitic therapeutics in the near future. Results showing their efficacy in murine infection models would seal the deal.

      Acknowledgments

      The author thanks Drs. Joyce Huang and Dave Diller for the various steroid chemical structures shown in Fig. 1 and Drs. Eli Mordechai and Martin Adelson for the many discussions pertaining to the manuscript. The author is grateful for the financial support of Genesis Biotechnology Group.

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