1921
Volume 81, Issue 4
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645

Abstract

There is an urgent need for new compounds for the drug development pipeline for treatment of patients with African sleeping sickness. One approach for identifying such compounds is by high throughput screening (HTS) of compound collections. For time and cost considerations, there is a need for the development of an assay that uses at least 384-well formats. To our knowledge, there are currently no viability assays for whole cell screening of trypanosomes in the 384-well plate format. We have developed and optimized an Alamar Blue viability assay in a 384-well format for bloodstream form strain 427 (BS427). The assay had a Z′ > 0.5 and tolerated a final dimethyl-sulfoxide concentration of 0.42%. Drug sensitivity was compared with those reported from previously developed 96-well methods and was found to be comparable. The sensitivity and cost benefit of the Alamar Blue assay make it an excellent candidate for HTS application.

Loading

Article metrics loading...

/content/journals/10.4269/ajtmh.2009.09-0015
2009-10-01
2017-09-23
Loading full text...

Full text loading...

/deliver/fulltext/14761645/81/4/0810665.html?itemId=/content/journals/10.4269/ajtmh.2009.09-0015&mimeType=html&fmt=ahah

References

  1. World Health Organization, 2006. Human African trypanosomiasis (sleeping sickness): epidemiological update. Wkly Epidemiol Rec 81: 71–80.
  2. Moore AC, 2005. Prospects for improving African trypanosomiasis chemotherapy. J Infect Dis 191: 1793–1795.
  3. Brun R, Schumacher R, Schmid C, Kunz C, Burri C, 2001. The phenomenon of treatment failures in human African trypanosomiasis. Trop Med Int Health 6: 906–914.
  4. Chappuis F, Udayraj N, Stietenroth K, Meussen A, Bovier PA, 2005. Eflornithine is safer than melarsoprol for the treatment of second-stage Trypanosoma brucei gambiense human African trypanosomiasis. Clin Infect Dis 41: 748–751.
  5. Pink R, Hudson A, Mouries MA, Bendig M, 2005. Opportunities and challenges in antiparasitic drug discovery. Nat Rev Drug Discov 4: 727–740.
  6. Mackey ZB, Baca AM, Mallari JP, Apsel B, Shelat A, Hansell EJ, Chiang PK, Wolff B, Guy KR, Williams J, McKerrow JH, 2006. Discovery of trypanocidal compounds by whole cell HTS of Trypanosoma brucei. Chem Biol Drug Des 67: 355–363.
  7. Martyn DC, Jones DC, Fairlamb AH, Clardy J, 2007. High-throughput screening affords novel and selective trypanothione reductase inhibitors with anti-trypanosomal activity. Bioorg Med Chem Lett 17: 1280–1283.
  8. O’Brien J, Wilson I, Orton T, Pognan F, 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267: 5421–5426.
  9. Bienen EJ, Webster P, Fish WR, 1991. Trypanosoma(Nannomonas) congolense: changes in respiratory metabolism during the life cycle. Exp Parasitol 73: 403–412.
  10. Raz B, Iten M, Grether-Buhler Y, Kaminsky R, Brun R, 1997. The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro. Acta Trop 68: 139–147.
  11. Lanteri CA, Stewart ML, Brock JM, Alibu VP, Meshnick SR, Tidwell RR, Barrett MP, 2006. Roles for the Trypanosoma brucei P2 transporter in DB75 uptake and resistance. Mol Pharmacol 70: 1585–1592.
  12. Stewart ML, Boussard C, Brun R, Gilbert IH, Barrett MP, 2005. Interaction of monobenzamidine-linked trypanocides with the Trypanosoma brucei P2 aminopurine transporter. Antimicrob Agents Chemother 49: 5169–5171.
  13. Merschjohann K, Steverding D, 2006. In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators. Kinetoplastid Biol Dis 5: 3.
  14. Rifkin MR, 1978. Identification of the trypanocidal factor in normal human serum: high density lipoprotein. Proc Natl Acad Sci USA 75: 3450–3454.
  15. Damper D, Patton CL, 1976. Pentamidine transport and sensitivity in Brucei-group trypanosomes. J Protozool 23: 349–356.
  16. Hoet S, Opperdoes F, Brun R, Adjakidje V, Quetin-Leclercq J, 2004. In vitro antitrypanosomal activity of ethnopharmacologically selected Beninese plants. J Ethnopharmacol 91: 37–42.
  17. Klenke B, Stewart M, Barrett MP, Brun R, Gilbert IH, 2001. Synthesis and biological evaluation of s-triazine substituted polyamines as potential new anti-trypanosomal drugs. J Med Chem 44: 3440–3452.
  18. Zhang JH, Chung TD, Oldenburg KR, 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4: 67–73.
  19. Iversen PW, Eastwood BJ, Sittampalam GS, Cox KL, 2006. A comparison of assay performance measures in screening assays: signal window, Z′ factor, and assay variability ratio. J Biomol Screen 11: 247–252.
  20. Sui Y, Wu Z, 2007. Alternative statistical parameter for high-throughput screening assay quality assessment. J Biomol Screen 12: 229–234.
  21. Hirumi H, Hirumi K, 1989. Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J Parasitol 75: 985–989.
  22. Seethala R, Fernandes PB, Ebooks Corporation, 2001. Handbook of Drug Screening. New York: Marcel Dekker, 15.
  23. Graham SV, Wymer B, Barry JD, 1998. Activity of a trypanosome metacyclic variant surface glycoprotein gene promoter is dependent upon life cycle stage and chromosomal context. Mol Cell Biol 18: 1137–1146.
  24. Even MS, Sandusky CB, Barnard ND, 2006. Serum-free hybridoma culture: ethical, scientific and safety considerations. Trends Biotechnol 24: 105–108.
  25. Bohets HH, Nouwen EJ, De Broend ME, Dierickx PJ, 1994. Effects of foetal calf serum on cell viability, cytotoxicity and detoxification in the two kidney-derived cell lines LLC-PK 1 and MDCK. Toxicol In Vitro 8: 559–561.
  26. Hamid R, Rotshteyn Y, Rabadi L, Parikh R, Bullock P, 2004. Comparison of Alamar blue and MTT assays for high throughput screening. Toxicol In Vitro 18: 703–710.
http://instance.metastore.ingenta.com/content/journals/10.4269/ajtmh.2009.09-0015
Loading
/content/journals/10.4269/ajtmh.2009.09-0015
Loading

Data & Media loading...

  • Received : 12 Jan 2009
  • Accepted : 07 Jul 2009

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error