A key enzyme in nucleic acid biosynthesis : The 5’-cytosolic nucleotidase (cN-II) as new drug target – cN-II Focus

Several approaches have been proposed:
1) the search for substrate analogues (nucleoside 5'-monophosphate derivatives or nucleotides); it has been initiated through the synthesis of chemically and enzymatically stable compounds such as phosphonate nucleoside analogues. Using the structural data related to the cN-II and available in the litterature, molecular modelling experiments will allow to rationalize and optimize the lead-compounds.
2) the use of a fragment based drug-design (FBDD), this approach is based on the screening of a fragment-library on the purified protein using NMR. The identification of fragments (of low molecular weigth and chemical complexity) is performed on the basis of their affinity with the targeted protein (in the mM range). These lasts are considered as basic scaffolds that must be combined or optimized to give rise to lead compounds, and further modified to obtain drug-like derivatives.
3) the in silico screening of commercially available libraries.

All these approaches being complementary, it may allow the identification of both allosteric or competitive inhibitors with no-related chemical structure.

Our main objective is to design and to study inhibitors of this enzyme in order to potentiate the efficiency of nucleoside analogues currently used in clinic. The rational design of cN-II inhibitors, such as allosteric effectors and substrate analogues, i.e. purine mononucleotides, is performed using molecular modelling and x-ray crystallography data. In the mean time, we also performed in silico screening of libraries and developed a novel NMR-fragment based drug-design approach. The screening and identification of potential inhibitors is carried out in vitro using the recombinant purified enzyme and biological activity is determined in cell culture (alone or in association with nucleoside analogues).
We also plan to study the implication of cN-II in the activation and/or the catabolism of cytotoxic nucleoside analogues and the impact of cN-II inhibition on the cellular metabolism of nucleotide. Therefore, an analytical method (for quantification and detection of inosine, the product released during the hydrolysis of IMP by cN-II) has been settled.

Based on these results from our laboratory and other internationally recognized groups our working hypothesis is that cN-II 5’-nucleotidase may be considered as a new target in chemotherapy, especially its inhibition would potentiate the effect of cytotoxic nucleoside analogues. These results suggest that the inhibition of cN-II in human cancer cells could either directly induce cell death or sensitize these cells to the activity of nucleoside analogues. Given the crucial role of nucleoside analogues in the treatment of malignant haematological diseases and the development of new drugs belonging to this family, an enzyme inhibitor inducing increased activity or restoring activity of the nucleoside analogues would have tremendous economical and social interest.

Review: Therapeutic perspectives for cN-II in cancer, Lars Petter Jordheim & Laurent Chaloin in Current Medicinal Chemistry, Vol 20, 2013, in press.

Nucleoside analogues are widely used as antiviral and anticancer agents. These compounds mimic physiological metabolites and interfere with key steps during nucleic acids biosynthesis and/or in cancer cell proliferation, respectively. The development of novel cytotoxic agents is currently limited by the appearance of resistance mechanisms mainly consisting in altered nucleotide metabolism. Among, the five cytosolic human nucleotidases involved in the regulation of cellular nucleotide pools, the over expression of 5’-nucleotidase II (cN-II) has been shown to be a predictive and non-favourable factor for patient survival when treated by cytarabine (a well-know nucleoside analogue used to treat leukaemia). Thus, the involvement of cN-II as a new resistance mechanism to cytotoxic nucleoside analogues is suspected.

The main objective of our proposal is to design and to study inhibitors of this enzyme in order to potentiate the efficiency of nucleoside analogues currently used in clinic.
The rational design of cN-II inhibitors, such as allosteric effectors and substrate analogues, i.e. purine mononucleotides, will be performed using molecular modelling, X-ray crystallography and a novel NMR-fragment based drug-design approach.
The screening of nucleotidic analogues as well as structurally unrelated inhibitors will be carried out in vitro using the recombinant purified enzyme and biological activity of the lead compounds (alone or in association with nucleoside analogues) will be determined in cell culture and in vivo.
In the meantime, we will also study: (i) the specificity of our derivatives towards the other members of the nucleotidases’ family, (ii) the implication of cN-II in the activation and/or the catabolism of cytotoxic nucleoside analogues and finally (iii) the impact of cN-II inhibition on the cellular metabolism of nucleotide.

Through the use of selective inhibitors of cN-II, our plural-disciplinary project involving chemistry, crystallography, molecular modelling, enzymology and biological evaluation will give essential data on the cellular role and function of the 5’-nucleotidase cN-II.