Departments of 1 Molecular Microbiology, and 3 Medicine, Washington University School of Medicine, St Louis, MO 63110; 2 Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, USA
Received 6 July 2003; returned 1 December 2003; revised 5 January 2004; accepted 13 April 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: We tested the effects of cyclopropyl (CPC) and cyclobutyl (CBC) carbinols on the survival of Entamoeba histolytica trophozoites and on the enzymatic activities of E. histolytica alcohol dehydrogenase 2 (EhADH2), a crucial enzyme in the amoebic fermentation pathway.
Results: At 72 h, the estimated 50% inhibitory concentrations of CPC and CBC were 38.9 and 11.2 µM, respectively. The EhADH2 alcohol and aldehyde dehydrogenase activities were inhibited by 1.82 µM CPC and 0.89 µM CBC in vitro.
Conclusions: CPC and CBC are expected to be non-toxic to humans at the concentrations required to eliminate E. histolytica trophozoites. Similarities between EhADH2 and the Giardia lamblia AdhE enzyme indicate that CPC and CBC could be effective drugs for treatment of both amoebiasis and giardiasis.
Keywords: bifunctional proteins , glycolytic pathways , eukaryotic parasites , cycloalkanols , alcohol dehydrogenase E
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Successful complementation of a mutant Escherichia coli strain (SHH31adhE) with recombinant EhADH212,13 provides an ideal system for studying potential inhibitors of EhADH2. In this study, we show that the cycloalkanol compounds cyclopropyl (CPC) and cyclobutyl (CBC) carbinols inhibit the growth and survival of E. histolytica trophozoites. We also demonstrate that these agents specifically affect the ALDH and ADH activities of the recombinant EhADH2.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The different phylogenetic origin and unique structural characteristics of EhADH2 with respect to the human dehydrogenases also suggest that CPC and CBC would bind specifically to this enzyme and probably not to the human ADH and ALDH homologues. We have hypothesized that EhADH2 originated from the fusion of ancestral ADH and ALDH microbial proteins, diverging functionally and structurally from classical unifunctional ADHs and ALDHs.11 The anaerobic EhADH2 uses iron as the catalytic ion and shares the co-factor binding site for the ADH and ALDH activities.11 CPC and CBC apparently have strong affinity for this shared binding site. In contrast, humans possess two structurally independent ADH and ALDH aerobic enzymes; ADH is zinc dependent and ALDH requires no catalytic ion. Based on these differences between the amoebic and the human dehydrogenases, we predict little affinity of cycloalkanols for the mammalian enzymes.
Although CPC and CBC can be toxic to humans at millimolar concentrations, our EIC50 data indicate that, if administered at micromolar concentrations, these or similar compounds could be used safely as anti-amoebic agents. As anaesthetics, cycloalkanols act on the cytochrome P450 enzyme family and on neuronal conduction mechanisms (nicotinic acetylcholine receptor)18,2023 at much higher concentrations than the ones we used to inhibit trophozoite growth (Table 1). In a previous report,18 the anaesthetic EC50 value for cyclobutyl methanol (a CBC analogue) on vertebrates was 5.4 mM, 480 times higher than the CBC EIC50 value reported here (Table 1), and 6000-fold the concentration we needed to inhibit the ADH activity in vitro (Table 2). Mammalian toxicity data on the effects of n-alkanols on ion channels indicate that at least 140 mM cyclopropanemethanol and 14 mM cyclobutanemethanol were necessary to inhibit the nicotinic acetylcholine receptor in vitro.22 These values are 3600- and 1250-fold the concentrations used in our study to inhibit 50% of amoebic trophozoite growth by the analogues CPC and CBC, respectively (Table 1). A survey revealed that 1-aminocyclopropanol, an aldehyde dehydrogenase inhibitor, had no inhibitory effect on rat brain tryptophan hydrolase activity in vitro.21 A recent in vivo study showed that methyl isobutyl carbinol (MIBC) had low potential for toxicity in male rats, based on the plasma levels found after 12 h of a single 5 mmol/kg oral dose of MIBC. Neither mortality nor clinical signals of toxicity were observed in the animals.24 In summary, no study has reported any harmful effect of these or similar compounds on vertebrate dehydrogenases.
In conclusion, our results suggest that CPC and CBC can be used safely and effectively for the treatment of amoebiasis. These compounds are expected to be non-toxic to humans at the concentrations required to eliminate E. histolytica trophozoites. As anti-amoebic agents, CPC and CBC appear to specifically target EhADH2, while as anesthetics they act on P450 oxygenases and neuronal receptors. Similarities between EhADH2 and the Giardia lamblia AdhE enzyme17,25 indicate that CPC and CBC could be effective drugs for the treatment of both amoebiasis and giardiasis.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Walsh, J. A. (1986). Problems in recognition and diagnosis of amoebiasis: estimation of the global magnitude of morbidity and mortality. Reviews in Infectious Diseases 8, 22838.[ISI][Medline]
3 . Goldman, P., Koch, R. L., Yeung, T. C. et al. (1986). Comparing the reduction of nitroimidazoles in bacteria and mammalian tissues and relating it to biological activity. Biochemical Pharmacology 35, 4351.[CrossRef][ISI][Medline]
4 . Knight, R. C., Skolioowski, I. M. & Edwards, D. I. (1978). The interaction of reduced metronidazole with DNA. Biochemical Pharmacology 27, 208993.[CrossRef][ISI][Medline]
5 . Koch, C. J., Lord, E. M., Shapiro, I. M. et al. (1997). Imaging hypoxia and blood flow in normal tissues. Advances in Experimental Medicine and Biology 428, 58593.[ISI][Medline]
6 . Kock, R. L., Beaulieu, B. B., Jr, Chrystal, E. J. T. et al. (1981). A metronidazole metabolite in human urine and its risk. Science 211, 398400.[ISI][Medline]
7 . Reeves, R. E. (1984). Metabolism of Entamoeba histolytica Schaudinn, 1903. Advances in Parasitology 23, 10542.[ISI][Medline]
8 . Lo, H. & Reeves, R. E. (1978). Pyruvate-to-ethanol pathway in Entamoeba histolytica. Biochemical Journal 171, 22530.[ISI][Medline]
9 . Yang, W., Li, E., Kairong, T. et al. (1994). Entamoeba histolytica has an alcohol dehydrogenase homologous to the adhE gene product of Escherichia coli. Molecular and Biochemical Parasitology 64, 25360.[CrossRef][ISI][Medline]
10 . Espinosa, A., Wang, L., Li, E. et al. (1997). Expression of the alcohol dehydrogenase (ADH) domain of Entamoeba histolytica EhADH2 enzyme. Archives of Medical Research 28, S7881.
11
.
Espinosa, A., Yang, L., Zhang, Z. et al. (2001). The bifunctional Entamoeba histolytica 2 (EhADH2) protein is necessary for amoebic growth and survival and requires an intact C-terminal domain for both alcohol dehydrogenase and aldehyde dehydrogenase activity. Journal of Biological Chemistry 276, 2013643.
12 . Goodlove, P. E., Cunningham, P. R., Parker, J. et al. (1989). Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli. Gene 85, 20914.[CrossRef][ISI][Medline]
13
.
Yong, T., Li, E., Clark, D. et al. (1996). Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new anti-amoebic drugs. Proceedings of the National Academy of Sciences, USA 93, 64649.
14
.
Kessler, D., Herth, W. & Knappe, J. (1992). Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli. Journal of Biological Chemistry 267, 180739.
15 . Kessler, D., Leibrecht, I. & Knappe, J. (1991). Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE. FEBS Letters 281, 5963.[CrossRef][ISI][Medline]
16 . Reid, M. F. & Fewson, C. A. (1994). Molecular characterization of microbial alcohol dehydrogenases. Critical Reviews in Microbiology 20, 1356.[ISI][Medline]
17
.
Samuelson, J. (1999). Why metronidazole is active against both bacteria and parasites. Antimicrobial Agents and Chemotherapy 43, 153341.
18 . LaBella, F. S., Chen, Q. M., Stein, D. et al. (1997). The site of general anesthesia and cytochrome P450 oxygenases: similarities defined by straight chain and cyclic alcohols. British Journal of Pharmacology 120, 115864.[Abstract]
19 . Lancaster, F. E. (1992). Alcohol, nitric oxide, and neurotoxicity: is there a connection? A review. Alcohol Clinical & Experimental Research 6, 53941.
20 . Sherry, B. & Abeles, R. H. (1985). Mechanism of action of methanol oxidase, reconstitution of methanol oxidase with 5-deazaflavin, and inactivation of methanol oxidase by cyclopropanol. Biochemistry 24, 2594605.[ISI][Medline]
21 . Nilsson, G. E. & Tottmar, O. (1987). Effects of biogenic aldehydes and aldehyde dehydrogenase inhibitors on rat brain tryptophan hydrolase activity in vitro. Brain Research 409, 3749.[CrossRef][ISI][Medline]
22 . Wood, S. C., Hill, W. A. & Miller, K. W. (1993). Cycloalkanemethanols discriminate between volume- and length-dependent loss of activity of alkanols at the torpedo nicotinic acetylcholine receptor. Molecular Pharmacology 44, 121926.[Abstract]
23 . Raines, D. E., Claycomb, R. J., Scheller, M. et al. (2001). Nonhalogenated alkane anesthetics fail to potentiate agonistic actions on two ligand-gated ion channels. Anesthesiology 95, 4707.[ISI][Medline]
24 . Gingell, R., Regnier, J. F., Wilson, D. M. et al. (2003). Comparative metabolism of methyl isobutyl carbinol and methyl isobutyl ketone in male rats. Toxicology Letters 136, 199204.[CrossRef][ISI][Medline]
25 . Wang, D. M. (2000). Role of alcohol dehydrogenase E (ADHE) in the energy metabolism of Giardia lamblia. Molecular and Biochemical Parasitology 109, 2536.[CrossRef][ISI][Medline]
|