Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase.
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.
Buckner
F S
FS
Department of Medicine, University of Washington, Seattle 98195, USA.
Verlinde
C L
CL
La Flamme
A C
AC
Van Voorhis
W C
WC
D016475:3T3 Cells; D000818:Animals; D015139:Blotting, Southern; D046508:Culture Techniques; D004353:Drug Evaluation, Preclinical; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D014344:Trypanocidal Agents; D001616:beta-Galactosidase