INHIBITOR
SCREENING
Contract Research Services

Aim: Screening of compounds for their abilities to inhibit human PNP in vitro. Determination of the inhibition kinetics of a given compound on human recombinant PNP and measurement of its IC₅₀ value.
PNP enzyme: PNP enzyme used in the assay is a human recombinant PNP, cloned by NOVOCIB from human cells, expressed in E. coli, and produced and purified by NOVOCIB.
Enzyme QC: The PNP enzyme purity is controlled before every assay by SDS-PAGE. Enzyme activity is systematically controlled before performing any assay.
Replicate assays: One point is defined as a well per compound and per concentration tested. PNP in vitro assays are usually performed in duplicate (2 wells per compound and per concentration). Triplicates are available upon request.
PNP inhibition control: Ganciclovir (GCV), dissolved in DMSO, is used as positive control for PNP inhibition. Other positive control than GCV can be used if available. Both negative and positive controls are done in duplicate.
Enzymatic Reaction: The assays are performed on 96-well microplate, at 37°C, in 200µl of reaction buffer.
Procedure: Every assay, from one to 90 points, is done with one negative control, containing DMSO with no inhibitor, and:
- For Screening Assays: 2 positive controls containing GCV as a PNP inhibitor at final concentrations of 250µM and 10mM
- For Inhibition Kinetics (IC₅₀): 8 or 12 positive controls containing GCV at different concentrations, equally spaced by 3-fold dilutions to cover a 3.3-log wide range, or by 2-fold dilutions to cover 5-log wide range, respectively.
Controls are done in duplicate. If an additional microplate is needed, it includes the complete set of controls (in duplicate). Additional concentrations of inhibitor can be tested.

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For the Kinetics Analysis, IC50 is determined by plotting the residual activity as a function of inhibitor concentration. IC50 is then calculated using a standard four-parameter nonlinear regression analysis.

Plotting: As far as possible, the inhibitor concentration range is determined in order to get *: • half of the data points +/- 1 above the IC50 value or half +/- 1 below • well-defined top and bottom plateau values, at least within a 15% margin of theoretical values. * Abiding by these constraints depends on the availability of information about the compound before starting the assay. When the results of the assay do not meet two of these three constraints, whereas PNP inhibition by the compound is demonstrated, an additional assay can be performed with ad hoc alterations of the procedure (e.g. inhibitor concentration range, additional points, substrate concentration…)

(Optional) Confirmation by HPLC: For every positive assay, an HPLC (Agilent 1100 series) control of PNP inhibition can be performed by measuring inosine (IR) and Hypoxanthine (Hx) concentrations in the assay and in comparison with negative and positive controls.

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Metabolic function
Purine Nucleoside Phosphorylase (PNP) is involved in salvage pathway of the purine metabolism.

Catalytic activity
Purine Nucleoside Phosphorylase (PNP) is involved in salvage pathway of the purine metabolism. PNP catalyzes the cleavage of the glycosidic bond of ribo- or deoxyribonucleosides, in the presence of inorganic phosphate as a second substrate, to generate the purine base and ribose- or deoxyribose-1-phosphate. The reaction is reversible for natural substrates:
Therapeutic potential of PNP inhibitors
PNP deficiency leads to T-lymphocytopenia, usually with no apparent effects on B-lymphocyte function. These symptoms suggest possible chemotherapeutic applications of potent inhibitors of PNP, as selective immunosuppressive agents, to treat T-cell leukemias or T-cell-mediated autoimmune diseases, such as lupus erythematosus and rheumatoid arthritis1^(1,2).
The decrease in plasma and urine levels of urate is an additional symptom of PNP deficiency. PNP inhibitors may contribute to treat hyperuricemic states, such as secondary or xanthine gout.
Some PNP inhibitors have undergone clinical trials for the treatment of cutaneous T-cell lymphoma, acute lymphoblastic leukemia (ALL), HIV infections, and psoriasis.

BCX-34 (Peldesine) BCX-1777 (Forodesine) Peldesine (BCX-34) was granted orphan drug status for the treatment of T-cell lymphoma and reached phase III as an immunomodulator for inflammatory autoimmune diseases. It has recently entered clinical trial for HIV infections(3). Forodesine (BCX-1777) has US orphan drug status for the treatment of T-cell non-Hodgkin's lymphoma, including cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukaemia (CLL) and related leukaemias, including T-cell prolymphocytic leukaemia (PLL), adult T-cell leukaemia and hairy cell leukaemia, and for the treatment of acute lymphocytic leukaemia (ALL). Forodesine was also designed Orphan drug in Europe for ALL in December 2006, and for CTCL in February 2007(4).

PNP inhibitors are also investigated to prevent the cleavage, and the resulting deactivation of Nucleoside Analogues by PNP.

Note: Protozoan parasites are auxotrophic for purine and have their own PNPs which have specific activities and properties that differ from the human PNP. Protozoan parasites PNPs are considered to be reasonable target against infection (e.g. Plasmodium falciparum)⁽⁵⁾.

PNP, a threat for therapeutic efficacy of Nucleoside Analogues
In vivo, phosphorolysis is highly favoured over purine nucleoside synthesis and is coupled with two additional enzymatic reactions: oxidation of the liberated purine base by xanthine oxidase (XO) and its phosphoribosylation by hypoxanthine-guanine phosphoribosyltransferase (HGPRT)⁽⁶⁾. Thus, PNP plays a key role in the salvage pathway of the purine metabolism, enabling the cell to utilize purine bases recovered from metabolized purine ribo- and deoxyribonucleosides to synthesize purine nucleotides.
This phosphorolysis reaction of purine nucleosides by PNP has a direct impact on the therapeutic efficacy of Nucleoside Analogues. Antitumour or antiviral nucleoside analogues are likely to be cleaved by PNP before being phosphorylated by the cell nucleoside kinases and converted to the active nucleotide form. For instance, 2',3'-dideoxyguanosine (ddG)⁽⁷⁾, 9-β-D-arabinofuranosyl guanine (AraG)⁽⁸⁾ as well as one of its produg, Nelarabine (Arranon^®, GSK)⁽⁹⁾, which is intracellularly converted to AraG by Adenosine deaminase (ADA), are PNP resistant nucleoside analogues, whereas 2',3'-dideoxyinosine (ddI)⁽¹⁰⁾ is easily cleaved in vivo by PNP.
Since acyclonucleoside analogues are particularly resistant to cleavage by PNP though phosphorylated by viral thymidine kinases (TK), they are generally considered as excellent candidates as antiviral agents (e.g. aciclovir, ganciclovir)⁽¹¹⁾.

Note that Ganciclovir is not only PNP resistant, but is also a PNP inhibitor.

PNP, a tool for enzymatic synthesis of Nucleoside Analogues
The reversible reaction catalyzed by PNP can be favorably exploited to synthesize nucleoside analogues, especially when chemical synthesis is difficult to prepare and / or gives low yields.
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References (with links to PubMed) 1. S. Hikishima et al. (2007): Synthesis and biological evaluation of 9-deazaguanine derivatives connected by a linker to difuoromethylene phosphonic acid as multi-substrate analogue inhibitors of PNP Bioorg. Med. Chem. Lett. 17(15) 4173–4177B 2. T. Yokomatsu et al. (1999): Synthesis and biological evaluation of 1,1-difluoro-2-(tetrahydro-3-furanyl)ethylphosphonic acids possessing a N9-purinylmethyl functional group at the ring, a new class of inhibitors for purine nucleoside phosphorylases Bioorg. Med. Chem. Lett. 9(19) 2833-2836 3. ClinicalTrials.gov (NIH) 4. Medscape 5. K. Chaudhary et al. (2006): Toxoplasma gondii Purine Nucleoside Phosphorylase Biochemical Characterization, Inhibitor Profiles, and Comparison with the Plasmodium falciparum Ortholog J. Biol. Chem. 281(35), 25652-25658 6. A. Bzowska et al. (2000): Purine nucleoside phosphorylases: properties, functions, and clinical aspects Pharmacol. Ther. 88(3), 349-425 7. V. Gandhi et al. (1995): Cytotoxicity, metabolism, and mechanisms of action of 2',2'-difluorodeoxyguanosine in Chinese hamster ovary cells Cancer Res. 55(7), 1517-1524 8. L. C. Gravatt et al. (1993): Efficacy and toxicity of 9-beta-D-arabinofuranosylguanine (araG) as an agent to purge malignant T-cells from murine bone marrow: application to an in vivo T-cell leukemia model Leukemia 7(8), 1261-1267 9. C. U. Lambe et al. (1995): 6-Amino-6-methoxypurine arabinoside: an agent for T-cell malignancies Cancer Res. 55(15), 3352-3356 10. M. Weibel (1994): Potentiating effect of {2[2[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]ethenyl} phosphonic acid (MDL 74,428), a potent inhibitor of purine nucleoside phosphorylase, on the antiretroviral activities of 2',3'-dideoxyinosine combined to ribavirin in mice Biochem. Pharmacol. 48, 245-252 11. D. Shugar (1999): Viral and host-cell protein kinases: enticing antiviral targets, and relevance of nucleoside, and viral thymidine, kinases Pharmacol. Ther. 82(2-3), 315-335