Comparative complement selection in bacteria enables screening for lead compounds targeted to a purine salvage enzyme of parasites

Abstract

Expression plasmids encoding the hypoxanthine phosphoribosyltransferases (HPRTs) of Plasmodium falciparum, Schistosoma mansoni, Tritrichomonas foetus, and Homo sapiens were subcloned into genetically deficient Escherichia coli that requires complementation by the activity of a recombinant HPRT for growth on semidefined medium. Fifty-nine purine analogs were screened for their abilities to inhibit the growth of these bacteria. Several compounds that selectively altered the growth of the bacteria complemented by the malarial, schistosomal, or tritrichomonal HPRT compared with the growth of bacteria expressing the human enzyme were identified. These results demonstrate that the recombinant approach to screening compounds by complement selection in a comparative manner provides a rapid and efficient method for the identification of new lead compounds selectively targeted to the purine salvage enzymes of parasites.

Hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi as a target for structure-based inhibitor design: crystallization and inhibition studies with purine analogs

“…For the bacterial screening assays, the recombinant trypanosomal and human HPRTs (encoded in plasmids pTcPRT and pHum PRT, respectively) were used to complement the genetic deficiencies of the SØ609 strain of E. coli (21). When grown on selective medium supplemented with guanine, these bacteria may grow only if they are complemented by an active recombinant HPRT (11). By this screening assay, purine analogs were tested for their ability to bind the human and trypanosomal HPRTs on the basis of their effects on the growth of bacteria complemented by each of the recombinant enzymes.…”

“…During the early stages of bacterial growth, when phosphate levels in the medium are high, expression of the recombinant enzyme relies on the leaky phoA promoter, which allows sufficient quantities of the HPRTs to be produced to complement the genetic deficiency of the bacteria (11). The levels of recombinant HPRT expression during the early stages of cell growth are predicted to be in the range of 0.005 to 0.01% of the total bacterial proteins (11). These early conditions probably provide a fairly accurate simulation of the concentration of HPRTs in cells in vivo.…”

“…Initially, a recombinant screening system was used to identify compounds that bind to the trypanosomal HPRT. This screening assay is based on a technique which uses the activities of recombinant HPRTs to complement genetic deficiencies of the host bacteria (11). Results of kinetic studies of these compounds in which purified recombinant human and trypanosomal enzymes are used, further support the results from the bacterial screening assays.…”

Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase

“…These are by de novo synthesis starting with simple precursor molecules and by salvage and recycling of the purine bases. However, in T. brucei there are no enzymes for de novo synthesis and this parasite relies solely on its salvage pathways91011 to make its purine nucleoside monophosphates. The T. brucei genome project12 has identified many of the enzymes expected to play key roles in the recycling and salvage of purine bases and nucleosides.…”

Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants

“…Introduction. Antimetabolites are extensively used as drugs and pesticides and are widely studied as «lead» compounds in «drug dcsign» programs [1 ] (for rev. see [2,3]).…”