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 ce lls that express the Escherichia coli beta-galactosidase gene, Transfe cted 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 i mmunosorbent 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 amphoterici n B were shown to inhibit the parasites at concentrations similar to t hose previously reported. Several compounds were tested that are inhib itors of glyceraldehyde-3-phosphate dehydrogenase of the related organ isms Leishmania mexicana and Trypanosoma brucei. One of these compound s, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 1 0 mu M 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 th e bloodstream form of the parasite under conditions used for storing b lood products, Thus, an assay with beta-galactosidase-expressing T. cr uzi greatly simplifies screening drugs for selective anti-T. cruzi act ivity, and three promising new compounds have been identified.