Nucleic acid synthesis and degradation are ongoing metabolic processes in most cells. The degradative processes lead to the release of free purines and the salvage pathway exists to recover them efficiently in a useful form.
Purine nucleoside phosphorylase (PNP) represents one of the key enzymes of the purine salvage pathway, which is considerably more energy-efficient than de novo pathway. Human PNP is overexpressed in T-cell leukemia, breast and colon cancer and during autoimmune diseases and PNP thus has been established as prospective target for drug design. Several hPNP inhibitors recently entered human clinical trials.
For many parasites and bacteria the purine salvage pathway is major or the only way to obtain purine nucleotides for the synthesis of nucleic acids. PNP enzymes from Plasmodium falciparum (PfPNP) and Mycobacterium tuberculosis (MtPNP) are thus potential targets for treatment of malaria and tuberculosis.
We are using X-ray crystallography in structure-based drug design of novel acyclic nucleotide analogues. Our goal is to design inhibitors with high affinity towards hPNP, PfPNP and MtPNP, respectively.
Enzymes were prepared by heterologous expression in E. coli and purified in high yields and purity necessary for crystallographic studies. Crystallization conditions for hPNP, PfPNP and MtPNP were identified through wide screening and optimization. Selected inhibitors were successfully co-crystallized with MtPNP and hPNP, diffraction data have been collected on BL14.1 at the BESSY II electron storage ring operated by the Helmholtz-Zentrum Berlin and crystal structures were determined at high resolution. The knowledge of binding of these inhibitors in the enzyme will further help with the designing of specific PNP inhibitors.