Metabolite-protein interactions define the output of metabolic pathways and regulate many cellular processes. Although diseases are often characterized by distortions in metabolic processes, efficient means to discover and study such interactions directly in cells have been lacking. A stringent implementation of proteome-wide Cellular Thermal Shift Assay (CETSA) was developed and applied to key cellular nucleotides, where previously experimentally confirmed protein-nucleotide interactions were well recaptured. Many predicted, but never experimentally confirmed, as well as novel protein-nucleotide interactions were discovered. Interactions included a range of different protein families where nucleotides serve as substrates, products, co-factors or regulators. In cells exposed to thymidine, a limiting precursor for DNA synthesis, both dose- and time-dependence of the intracellular binding events for sequentially generated thymidine metabolites were revealed. Interactions included known cancer targets in deoxyribonucleotide metabolism as well as novel interacting proteins. This stringent CETSA based strategy will be applicable for a wide range of metabolites and will therefore greatly facilitate the discovery and studies of interactions and specificities of the many metabolites in human cells that remain uncharacterized.

To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of “off target effects.” However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔTagg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design.

.2.8) catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates. Human HPRT deficiency as a result of genetic mutations is linked to both Lesch-Nyhan disease and gout. In the present study, we have characterized phosphoribosyltransferase domain containing protein 1 (PRTFDC1), a human HPRT homolog of unknown function. The PRTFDC1 structure has been determined at 1.7 Å resolution with bound GMP. The overall structure and GMP binding mode are very similar to that observed for HPRT. Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme. It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate. However, the catalytic efficiency (k cat ⁄ K m ) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT. This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT. In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base. The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway.

Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.

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