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. 1996 Nov;40(11):2592–2597. doi: 10.1128/aac.40.11.2592

Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase.

F S Buckner 1, C L Verlinde 1, A C La Flamme 1, W C Van Voorhis 1
PMCID: PMC163582  PMID: 8913471

Abstract

A new drug screening method was devised utilizing Trypanosoma cruzi cells that express the Escherichia coli beta-galactosidase gene. Transfected parasites catalyze a colorimetric reaction with chlorophenol red beta-D-galactopyranoside as substrate. Parasite growth in the presence of drugs in microtiter plates was quantitated with an enzyme-linked immunosorbent assay reader. The assay was performed with the mammalian form of T. cruzi that requires intracellular growth on a monolayer of fibroblast cells. To determine if selective toxicity to the parasites was occurring, the viability of the host cells in the drug was assayed with AlamarBlue. The drugs benznidazole, fluconazole, and amphotericin B were shown to inhibit the parasites at concentrations similar to those previously reported. Several compounds were tested that are inhibitors of glyceraldehyde-3-phosphate dehydrogenase of the related organisms Leishmania mexicana and Trypanosoma brucei. One of these compounds, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 10 microM in our assay. Two derivatives of this compound were identified with in vitro activity at even lower concentrations. In addition, the assay was modified for testing compounds for lytic activity against the bloodstream form of the parasite under conditions used for storing blood products. Thus, an assay with beta-galactosidase-expressing T. cruzi greatly simplifies screening drugs for selective anti-T. cruzi activity, and three promising new compounds have been identified.

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Selected References

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  1. Ajioka J., Swindle J. The calmodulin-ubiquitin associated genes of Trypanosoma cruzi: their identification and transcription. Mol Biochem Parasitol. 1993 Jan;57(1):127–136. doi: 10.1016/0166-6851(93)90250-2. [DOI] [PubMed] [Google Scholar]
  2. Berens R. L., Marr J. J., Steele da Cruz F. S., Nelson D. J. Effect of allopurinol on Trypanosoma cruzi: metabolism and biological activity in intracellular and bloodstream forms. Antimicrob Agents Chemother. 1982 Oct;22(4):657–661. doi: 10.1128/aac.22.4.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Campos R., Amato Neto V., Moreira A. A., de Souza H. B., Okumura M., Pinto P. L., Braz L. M., Silva M. F., Matsubara L. Avaliaço da atividade terapêutica do fluconazol na infecço aguda, experimental, pelo Trypanosoma cruzi. Rev Hosp Clin Fac Med Sao Paulo. 1992 Jul-Aug;47(4):174–175. [PubMed] [Google Scholar]
  4. Chiari E., de Oliveira A. B., Raslan D. S., Mesquita A. A., Tavares K. G. Screening in vitro of natural products against blood forms of Trypanosoma cruzi. Trans R Soc Trop Med Hyg. 1991 May-Jun;85(3):372–374. doi: 10.1016/0035-9203(91)90296-b. [DOI] [PubMed] [Google Scholar]
  5. Chung S. H., Gillespie R. D., Swindle J. Analyzing expression of the calmodulin and ubiquitin-fusion genes of Trypanosoma cruzi using simultaneous, independent dual gene replacements. Mol Biochem Parasitol. 1994 Jan;63(1):95–107. doi: 10.1016/0166-6851(94)90012-4. [DOI] [PubMed] [Google Scholar]
  6. Cover B., Gutteridge W. E. A primary screen for drugs to prevent transmission of Chagas's disease during blood transfusion. Trans R Soc Trop Med Hyg. 1982;76(5):633–635. doi: 10.1016/0035-9203(82)90228-0. [DOI] [PubMed] [Google Scholar]
  7. Croft S. L., Walker J. J., Gutteridge W. E. Screening of drugs for rapid activity against Trypanosoma cruzi trypomastigotes in vitro. Trop Med Parasitol. 1988 Jun;39(2):145–148. [PubMed] [Google Scholar]
  8. Cruz F. S., Marr J. J., Berens R. L. Prevention of transfusion-induced Chagas' disease by amphotericin B. Am J Trop Med Hyg. 1980 Sep;29(5):761–765. doi: 10.4269/ajtmh.1980.29.761. [DOI] [PubMed] [Google Scholar]
  9. Goad L. J., Berens R. L., Marr J. J., Beach D. H., Holz G. G., Jr The activity of ketoconazole and other azoles against Trypanosoma cruzi: biochemistry and chemotherapeutic action in vitro. Mol Biochem Parasitol. 1989 Jan 15;32(2-3):179–189. doi: 10.1016/0166-6851(89)90069-8. [DOI] [PubMed] [Google Scholar]
  10. Grunberg E., Beskid G., Cleeland R., DeLorenzo W. F., Titsworth E., Scholer H. J., Richle R., Brener Z. Antiprotozoan and antibacterial activity of 2-nitroimidazole derivatives. Antimicrob Agents Chemother (Bethesda) 1967;7:513–519. [PubMed] [Google Scholar]
  11. Hammond D. J., Cover B., Gutteridge W. E. A novel series of chemical structures active in vitro against the trypomastigote form of Trypanosoma cruzi. Trans R Soc Trop Med Hyg. 1984;78(1):91–95. doi: 10.1016/0035-9203(84)90184-6. [DOI] [PubMed] [Google Scholar]
  12. Hammond D. J., Croft S. L., Hogg J., Gutteridge W. E. A strategy for the prevention of the transmission of Chagas' disease during blood transfusion. Acta Trop. 1986 Dec;43(4):367–378. [PubMed] [Google Scholar]
  13. Hammond D. J., Hogg J., Gutteridge W. E. Trypanosoma cruzi: possible control of parasite transmission by blood transfusion using amphiphilic cationic drugs. Exp Parasitol. 1985 Aug;60(1):32–42. doi: 10.1016/s0014-4894(85)80020-5. [DOI] [PubMed] [Google Scholar]
  14. Hariharan S., Ajioka J., Swindle J. Stable transformation of Trypanosoma cruzi: inactivation of the PUB12.5 polyubiquitin gene by targeted gene disruption. Mol Biochem Parasitol. 1993 Jan;57(1):15–30. doi: 10.1016/0166-6851(93)90240-x. [DOI] [PubMed] [Google Scholar]
  15. Horvath A. E., Zierdt C. H. The effect of amphotericin B on Trypanosoma cruzi in vitro and in vivo. J Trop Med Hyg. 1974 Jul;77(7):144–149. [PubMed] [Google Scholar]
  16. La Flamme A. C., Buckner F. S., Swindle J., Ajioka J., Van Voorhis W. C. Trypanosoma cruzi: expression of interleukin-2 utilizing both supercoiled plasmids and linear DNAs. Exp Parasitol. 1996 Jun;83(1):159–163. doi: 10.1006/expr.1996.0061. [DOI] [PubMed] [Google Scholar]
  17. Polak A., Richle R. Mode of action of the 2-nitroimidazole derivative benznidazole. Ann Trop Med Parasitol. 1978 Feb;72(1):45–54. doi: 10.1080/00034983.1978.11719278. [DOI] [PubMed] [Google Scholar]
  18. Seeber F., Boothroyd J. C. Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii. Gene. 1996 Feb 22;169(1):39–45. doi: 10.1016/0378-1119(95)00786-5. [DOI] [PubMed] [Google Scholar]
  19. Tomich C. S., Kaytes P. S., Olsen M. K., Patel H. Use of lacZ expression to monitor transcription. Plasmid. 1988 Sep;20(2):167–170. doi: 10.1016/0147-619x(88)90022-4. [DOI] [PubMed] [Google Scholar]
  20. Van Voorhis W. C., Eisen H. Fl-160. A surface antigen of Trypanosoma cruzi that mimics mammalian nervous tissue. J Exp Med. 1989 Mar 1;169(3):641–652. doi: 10.1084/jem.169.3.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Verlinde C. L., Callens M., Van Calenbergh S., Van Aerschot A., Herdewijn P., Hannaert V., Michels P. A., Opperdoes F. R., Hol W. G. Selective inhibition of trypanosomal glyceraldehyde-3-phosphate dehydrogenase by protein structure-based design: toward new drugs for the treatment of sleeping sickness. J Med Chem. 1994 Oct 14;37(21):3605–3613. doi: 10.1021/jm00047a017. [DOI] [PubMed] [Google Scholar]
  22. de Castro S. L., Pinto M. C., Pinto A. V. Screening of natural and synthetic drugs against Trypanosoma cruzi. 1. Establishing a structure/activity relationship. Microbios. 1994;78(315):83–90. [PubMed] [Google Scholar]
  23. de-Castro S. L., Soeiro M. N., Higashi K. O., Meirelles M. N. Differential effect of amphotericin B on the three evolutive stages of Trypanosoma cruzi and on the host cell-parasite interaction. Braz J Med Biol Res. 1993 Nov;26(11):1219–1229. [PubMed] [Google Scholar]

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