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Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1997 Feb;41(2):379–384. doi: 10.1128/aac.41.2.379

A cytotoxicity assay for evaluation of candidate anti-Pneumocystis carinii agents.

M T Cushion 1, F Chen 1, N Kloepfer 1
PMCID: PMC163717  PMID: 9021195

Abstract

A series of over 60 agents representing several different classes of compounds were evaluated for their effects on the ATP pools of Pneumocystis carinii populations derived from immunosuppressed rats. A cytotoxicity assay based on an ATP-driven bioluminescent reaction was used to determine the concentration of agent which decreased the P. carinii ATP pools by 50% versus untreated controls (IC50). A ranking system based on the IC50 value was devised for comparison of relative responses among the compounds evaluated in the cytotoxic assay and for comparison to in vivo efficacy. With few exceptions, there was a strong correlation between results from the ATP assay and the performance of the compound in vivo. Antibiotics, with the exception of trimethoprim-sulfamethoxazole (TMP-SMX), were ineffective at reducing the ATP pools and were not active clinically or in the rat model of P. carinii pneumonia. Likewise, other agents not expected to be effective, e.g., antiviral compounds, did not show activity. Standard anti-P. carinii compounds, e.g., TMP-SMX, pentamidine, and dapsone, dramatically reduced ATP levels. Analogs of the quinone and topoisomerase inhibitor groups were shown to reduce ATP concentrations and hold promise for further in vivo investigation. The cytotoxicity assay provides a rapid assessment of response, does not rely on replicating organisms, and should be useful for assessment of structure-function relationships.

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Selected References

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  1. Allegra C. J., Kovacs J. A., Drake J. C., Swan J. C., Chabner B. A., Masur H. Activity of antifolates against Pneumocystis carinii dihydrofolate reductase and identification of a potent new agent. J Exp Med. 1987 Mar 1;165(3):926–931. doi: 10.1084/jem.165.3.926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banerji S., Wakefield A. E., Allen A. G., Maskell D. J., Peters S. E., Hopkin J. M. The cloning and characterization of the arom gene of Pneumocystis carinii. J Gen Microbiol. 1993 Dec;139(12):2901–2914. doi: 10.1099/00221287-139-12-2901. [DOI] [PubMed] [Google Scholar]
  3. Bartlett M. S., Edlind T. D., Lee C. H., Dean R., Queener S. F., Shaw M. M., Smith J. W. Albendazole inhibits Pneumocystis carinii proliferation in inoculated immunosuppressed mice. Antimicrob Agents Chemother. 1994 Aug;38(8):1834–1837. doi: 10.1128/aac.38.8.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartlett M. S., Eichholtz R., Smith J. W. Antimicrobial susceptibility of Pneumocystis carinii in culture. Diagn Microbiol Infect Dis. 1985 Sep;3(5):381–387. doi: 10.1016/0732-8893(85)90076-8. [DOI] [PubMed] [Google Scholar]
  5. Bartlett M. S., Marr J. J., Queener S. F., Klein R. S., Smith J. W. Activity of inosine analogs against Pneumocystis carinii in culture. Antimicrob Agents Chemother. 1986 Jul;30(1):181–183. doi: 10.1128/aac.30.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bartlett M. S., Queener S. F., Tidwell R. R., Milhous W. K., Berman J. D., Ellis W. Y., Smith J. W. 8-Aminoquinolines from Walter Reed Army Institute for Research for treatment and prophylaxis of Pneumocystis pneumonia in rat models. Antimicrob Agents Chemother. 1991 Feb;35(2):277–282. doi: 10.1128/aac.35.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boylan C. J., Current W. L. Improved rat model of Pneumocystis carinii pneumonia: induced laboratory infections in Pneumocystis-free animals. Infect Immun. 1992 Apr;60(4):1589–1597. doi: 10.1128/iai.60.4.1589-1597.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brun-Pascaud M., Fay M., Zhong M., Bauchet J., Dux-Guyot A., Pocidalo J. J. Use of fluoroquinolones for prophylaxis of murine Pneumocystis carinii pneumonia. Antimicrob Agents Chemother. 1992 Feb;36(2):470–472. doi: 10.1128/aac.36.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen F., Cushion M. T. Use of an ATP bioluminescent assay to evaluate viability of Pneumocystis carinii from rats. J Clin Microbiol. 1994 Nov;32(11):2791–2800. doi: 10.1128/jcm.32.11.2791-2800.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clarkson A. B., Jr, Brohn F. H. Trypanosomiasis: an approach to chemotherapy by the inhibition of carbohydrate catabolism. Science. 1976 Oct 8;194(4261):204–206. doi: 10.1126/science.986688. [DOI] [PubMed] [Google Scholar]
  11. Clarkson A. B., Jr, Sarić M., Grady R. W. Deferoxamine and eflornithine (DL-alpha-difluoromethylornithine) in a rat model of Pneumocystis carinii pneumonia. Antimicrob Agents Chemother. 1990 Sep;34(9):1833–1835. doi: 10.1128/aac.34.9.1833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clarkson A. B., Jr, Williams D. E., Rosenberg C. Efficacy of DL-alpha-difluoromethylornithine in a rat model of Pneumocystis carinii pneumonia. Antimicrob Agents Chemother. 1988 Aug;32(8):1158–1163. doi: 10.1128/aac.32.8.1158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Comley J. C., Mullin R. J., Wolfe L. A., Hanlon M. H., Ferone R. Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii. Antimicrob Agents Chemother. 1991 Oct;35(10):1965–1974. doi: 10.1128/aac.35.10.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Comtois R., Pouliot J., Vinet B., Gervais A., Lemieux C. Higher pentamidine levels in AIDS patients with hypoglycemia and azotemia during treatment of Pneumocystis carinii pneumonia. Am Rev Respir Dis. 1992 Sep;146(3):740–744. doi: 10.1164/ajrccm/146.3.740. [DOI] [PubMed] [Google Scholar]
  15. Cushion M. T., Ebbets D. Growth and metabolism of Pneumocystis carinii in axenic culture. J Clin Microbiol. 1990 Jun;28(6):1385–1394. doi: 10.1128/jcm.28.6.1385-1394.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cushion M. T. In vitro studies of Pneumocystis carinii. J Protozool. 1989 Jan-Feb;36(1):45–52. doi: 10.1111/j.1550-7408.1989.tb02691.x. [DOI] [PubMed] [Google Scholar]
  17. Cushion M. T., Kaselis M., Stringer S. L., Stringer J. R. Genetic stability and diversity of Pneumocystis carinii infecting rat colonies. Infect Immun. 1993 Nov;61(11):4801–4813. doi: 10.1128/iai.61.11.4801-4813.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Cushion M. T., Ruffolo J. J., Linke M. J., Walzer P. D. Pneumocystis carinii: growth variables and estimates in the A549 and WI-38 VA13 human cell lines. Exp Parasitol. 1985 Aug;60(1):43–54. doi: 10.1016/s0014-4894(85)80021-7. [DOI] [PubMed] [Google Scholar]
  19. Cushion M. T., Stanforth D., Linke M. J., Walzer P. D. Method of testing the susceptibility of Pneumocystis carinii to antimicrobial agents in vitro. Antimicrob Agents Chemother. 1985 Dec;28(6):796–801. doi: 10.1128/aac.28.6.796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Desjardins R. E., Casero R. A., Jr, Willet G. P., Childs G. E., Canfield C. J. Trypanosoma rhodesiense: semiautomated microtesting for quantitation of antitrypanosomal activity in vitro. Exp Parasitol. 1980 Oct;50(2):260–271. doi: 10.1016/0014-4894(80)90027-2. [DOI] [PubMed] [Google Scholar]
  21. Dykstra C. C., Tidwell R. R. Inhibition of topoisomerases from Pneumocystis carinii by aromatic dicationic molecules. J Protozool. 1991 Nov-Dec;38(6):78S–81S. [PubMed] [Google Scholar]
  22. Ferguson L. R., Sundberg R. J. Petite mutagenesis in Saccharomyces cerevisiae by a series of bis-cationic trypanocidal drugs. Antimicrob Agents Chemother. 1991 Nov;35(11):2318–2321. doi: 10.1128/aac.35.11.2318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fishman J. A., Queener S. F., Roth R. S., Bartlett M. S. Activity of topoisomerase inhibitors against Pneumocystis carinii in vitro and in an inoculated mouse model. Antimicrob Agents Chemother. 1993 Jul;37(7):1543–1546. doi: 10.1128/aac.37.7.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Frenkel J. K., Good J. T., Shultz J. A. Latent Pneumocystis infection of rats, relapse, and chemotherapy. Lab Invest. 1966 Oct;15(10):1559–1577. [PubMed] [Google Scholar]
  25. Griffiths D. E., Houghton R. L. Studies on energy-linked reactions: modified mitochondrial ATPase of oligomycin-resistant mutants of Saccharomyces cerevisiae. Eur J Biochem. 1974 Jul 1;46(1):157–167. doi: 10.1111/j.1432-1033.1974.tb03608.x. [DOI] [PubMed] [Google Scholar]
  26. Hughes W. T., Smith B. L. Efficacy of diaminodiphenylsulfone and other drugs in murine Pneumocystis carinii pneumonitis. Antimicrob Agents Chemother. 1984 Oct;26(4):436–440. doi: 10.1128/aac.26.4.436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kuzmits R., Rumpold H., Müller M. M., Schopf G. The use of bioluminescence to evaluate the influence of chemotherapeutic drugs on ATP-levels of malignant cell lines. J Clin Chem Clin Biochem. 1986 May;24(5):293–298. doi: 10.1515/cclm.1986.24.5.293. [DOI] [PubMed] [Google Scholar]
  28. Mordelet-Dambrine M., Danel C., Farinotti R., Urzua G., Barritault L., Huchon G. J. Influence of Pneumocystis carinii pneumonia on serum and tissue concentrations of pentamidine administered to rats by tracheal injections. Am Rev Respir Dis. 1992 Sep;146(3):735–739. doi: 10.1164/ajrccm/146.3.735. [DOI] [PubMed] [Google Scholar]
  29. Pesanti E. L., Cox C. Metabolic and synthetic activities of Pneumocystis carinii in vitro. Infect Immun. 1981 Dec;34(3):908–914. doi: 10.1128/iai.34.3.908-914.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pifer L. L., Woods D., Hughes W. T. Propagation of Pneumocystis carinii in Vero cell culture. Infect Immun. 1978 Apr;20(1):66–68. doi: 10.1128/iai.20.1.66-68.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Queener S. F., Bartlett M. S., Richardson J. D., Durkin M. M., Jay M. A., Smith J. W. Activity of clindamycin with primaquine against Pneumocystis carinii in vitro and in vivo. Antimicrob Agents Chemother. 1988 Jun;32(6):807–813. doi: 10.1128/aac.32.6.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sevin B. U., Peng Z. L., Perras J. P., Ganjei P., Penalver M., Averette H. E. Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing. Gynecol Oncol. 1988 Sep;31(1):191–204. doi: 10.1016/0090-8258(88)90293-4. [DOI] [PubMed] [Google Scholar]
  33. Smaldone G. C., Vinciguerra C., Morra L. Urine pentamidine as an indicator of lung pentamidine in patients receiving aerosol therapy. Chest. 1991 Nov;100(5):1219–1223. doi: 10.1378/chest.100.5.1219. [DOI] [PubMed] [Google Scholar]

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