4-Aminopyrazolo[3,4-d]pyrimidine (4-APP) as a novel inhibitor of the RNA and DNA depurination induced by Shiga toxin 1

Shiga toxin 1 (Stx1) catalyses the removal of a unique and specific adenine from 28S RNA in ribosomes (RNA-N-glycosidase activity) and the release of multiple adenines from DNA (DNA glycosylase activity). Added adenine behave s as an uncompetitive inhibitor of the RNA-N-glycosidase reaction binding m ore tightly to the Stx1-ribosome complex than to the free enzyme. Several p urine derivatives and analogues have now been assayed as inhibitors of Stx1 . Most of the compounds showed only minor differences in the rank order of activity on the two enzymatic reactions catalysed by Stx1. The survey highl ights the importance of the amino group in the 6-position of the pyrimidine ring of adenine, Shifting (2-aminopurine) or substituting (hypoxanthine, 6 -mercaptopurine, 6-methylpurine) the group greatly decreases the inhibitory power. The presence of a second ring, besides the pyrimidine one, is stric tly required. Substitution, by introducing an additional nitrogen, of the i midazole ring of adenine with triazole leads to loss of inhibitory power, w hile rearrangement of the nitrogen atoms of the ring from the imidazole to the pyrazole configuration greatly enhances the inhibitory power. Thus 4-am inopyrazolo[3,4-d]pyrimidine (4-APP), the isomer of adenine with the five-m embered ring in the pyrazole configuration, is by far the most potent inhib itor of both enzymatic reactions catalysed by Stx1. This finding opens pers pectives on therapeutic strategies to protect endothelial renal cells once endocytosis of Stx1 has occurred (haemolytic uraemic syndrome). In the RNA- N-glycosidase reaction 4-APP binds, as adenine, predominantly to the Stx1-r ibosome complex (uncompetitive inhibition), while inhibition of the DNA gly cosylase activity by both inhibitors is of the mixed type.