Inhibition of p-Aminobenzoate and Folate Syntheses in Plants and Apicomplexan Parasites by Natural Product Rubreserine

Djeneb Camara,Cordelia Bisanz,Caroline Barette,Jeroen Van Daele,Esmare Human,Bernice Barnard,Dominique Van Der Straeten,Christophe P Stove,Willy E Lambert,Roland Douce,Eric Maréchal,Lyn-Marie Birkholtz,Marie-France Cesbron-Delauw,Renaud Dumas,Fabrice Rébeillé

doi:10.1074/jbc.m112.365833

Abstract

Glutamine amidotransferase/aminodeoxychorismate synthase (GAT-ADCS) is a bifunctional enzyme involved in the synthesis of p-aminobenzoate, a central component part of folate cofactors. GAT-ADCS is found in eukaryotic organisms autonomous for folate biosynthesis, such as plants or parasites of the phylum Apicomplexa. Based on an automated screening to search for new inhibitors of folate biosynthesis, we found that rubreserine was able to inhibit the glutamine amidotransferase activity of the plant GAT-ADCS with an apparent IC(50) of about 8 μM. The growth rates of Arabidopsis thaliana, Toxoplasma gondii, and Plasmodium falciparum were inhibited by rubreserine with respective IC(50) values of 65, 20, and 1 μM. The correlation between folate biosynthesis and growth inhibition was studied with Arabidopsis and Toxoplasma. In both organisms, the folate content was decreased by 40-50% in the presence of rubreserine. In both organisms, the addition of p-aminobenzoate or 5-formyltetrahydrofolate in the external medium restored the growth for inhibitor concentrations up to the IC(50) value, indicating that, within this range of concentrations, rubreserine was specific for folate biosynthesis. Rubreserine appeared to be more efficient than sulfonamides, antifolate drugs known to inhibit the invasion and proliferation of T. gondii in human fibroblasts. Altogether, these results validate the use of the bifunctional GAT-ADCS as an efficient drug target in eukaryotic cells and indicate that the chemical structure of rubreserine presents interesting anti-parasitic (toxoplasmosis, malaria) potential.

Highlights

PABA biosynthesis is a potential target for antifolate drugs
Rubreserine inhibits glutamine amidotransferase (GAT)-ADC synthase (ADCS), an enzyme involved in pABA biosynthesis, and decreases the folate content in Arabidopsis and Toxoplasma
Glutamine amidotransferase/aminodeoxychorismate synthase (GAT-ADCS) is a bifunctional enzyme involved in the synthesis of p-aminobenzoate, a central component part of folate cofactors

Summary

Conclusion

Specific inhibition of pABA synthesis induces growth limitation of plants and apicomplexan parasites. Based on an automated screening to search for new inhibitors of folate biosynthesis, we found that rubreserine was able to inhibit the glutamine amidotransferase activity of the plant GAT-ADCS with an apparent IC50 of about 8 ␮M. JUNE 22, 2012 VOLUME 287 NUMBER 26 dii in human fibroblasts These results validate the use of the bifunctional GAT-ADCS as an efficient drug target in eukaryotic cells and indicate that the chemical structure of rubreserine presents interesting anti-parasitic (toxoplasmosis, malaria) potential. Searches of inhibitors for this class of enzymes were only achieved using prokaryotic systems They involved docking studies and design of chorismate analogous compounds [22,23,24,25], combinatorial chemistry approaches [26, 27], and a specific screening of a microorganism extract collection using growth inhibition of test bacteria as a marker of activity [28, 29]. We identified one compound exhibiting a Ki Ͻ 10 ␮M and measured the impact of this molecule on a plant (Arabidopsis thaliana) and two apicomplexan parasites (T. gondii and P. falciparum)

EXPERIMENTAL PROCEDURES

RESULTS

60 Ϯ 10 85 Ϯ 9 66 Ϯ 3 116 Ϯ 12 54 Ϯ 9 42 Ϯ 3 39 Ϯ 6

DISCUSSION