INHIBITOR
SCREENING
Contract Research Services

Aim: Screening of compounds for their abilities to inhibit human IMPDH II in vitro. Determination of the inhibition kinetics of a given compound on human recombinant IMPDH II and measurement of its IC₅₀ value.
Human IMPDH II: The IMPDH II enzyme used in the assays is a human recombinant IMPDH II, cloned by NOVOCIB from human cells, expressed in E. coli, and produced and purified by NOVOCIB
Enzyme QC: The IMPDH II 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. IMPDH in vitro assays are usually performed in duplicate (2 wells per compound and per concentration). Triplicates are available upon request.
IMPDH II inhibition control: Mycophenolic Acid (MPA), dissolved in DMSO, is used as positive control for IMPDH II inhibition. Other positive control than MPA 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 (KH₂PO₄ 0.1M, pH7.8, NAD 250µM, DTT 2mM, IMP 250µM)
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 MPA as an IMPDH inhibitor at final concentrations of 150nM and 10µM
- For Inhibition Kinetics (IC₅₀): 8 or 12 positive controls containing MPA at different concentrations, equally spaced by 3- or 2-fold dilutions respectively, to cover a 3.3-log wide range.
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 IMPDH II 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…)

(Optional) For every positive result of a Screening assay, a confirmation by HPLC (Agilent 1100 series) of IMPDH II inhibition can be performed upon request by measuring IMP, XMP, NAD and NADH concentrations in the assay and by comparison with negative and positive controls.

Catalytic activity
Inosine Monophosphate Dehydrogenase (IMPDH) converts inosine 5’-monophosphate (IMP) to xanthosine 5’- monophosphate (XMP) using NAD⁺ as a cofactor. The oxidation of IMP to XMP is considered as the pivotal step in the biosynthesis of guanine nucleotide, whose pool controls cell proliferation and many other major cellular processes⁽¹⁾. The decrease in guanine nucleotide resulting from IMPDH inhibition interrupts the nucleic acid synthesis in proliferating cells. The involvement of IMPDH in de novo guanine nucleotide biosynthesis makes IMPDH a crucial enzyme in cell proliferation and differentiation⁽²⁾. IMPDH is recognized as a validated target for several major therapeutic areas. IMPDH inhibitors are exploited as antiviral (e.g. ribavirine), antiparasitic, antimicrobial, antileukemic and immunosuppressive agents⁽²⁾. IMPDH Type II is the predominant isoform of the enzyme and is selectively expressed in proliferating cells, including lymphocytes and tumor cells⁽²⁾.
IMPDH in immunology
IMPDH is highly active in lymphocytes. It is a validated target to treat immunological diseases and to induce immunosuppression (CellCept^®, a mycophenolic acid (MPA) prodrug - Roche – CHF1.85 Bn as an immunosuppressive agent in 2006, orphan drug designation in 2006 for Myasthenia Gravis; CellCept^® reached positive results in Phase III trials in Lupus Nephritis). IMPDH is also recognized as an excellent target for the treatment of psoriasis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE)⁽³⁾.
IMPDH in oncology
IMPDH, and particularly Type II, which is overexpressed in tumor cells, is considered as a highly potent target for cancer chemotherapy^{(1, 2, 4, 5)}. Several IMPDH inhibitors are under development for the treatment of Acute and Chronic Myelogenous Leukemia (AML, CML)⁽⁶⁾ and other cancers (pancreas, colon, bladder…). Additionally, it has been shown that the use of IMPDH inhibitors counteracts the drug resistance⁽⁷⁾ that may appear in certain tumors. For instance, methotrexate resistance is directly related to the overexpression of IMPDH, whose inhibition restores the drug efficacy⁽⁸⁾. Combination with other anti-cancer drugs extends the potential application of IMPDH inhibitors.
Current development of IMPDH inhibitors
CellCept^®, ribavirin, mizoribine and tiazofurine are examples of currently used drugs that target IMPDH. Benzamide riboside, tiazofurine, MPA are under development in Phase II/III in leukemia: results are judged very encouraging⁽⁸⁾.
The IMPDH II atomic structure has been resolved and it provides a valuable background for further leads optimization⁽⁹⁾. Besides nucleosides analogues, NCEs have been identified as IMPDH inhibitors^{(10, 11, 12, 13, 14)} and enter development trials (e.g. AVN-944: Phase I in advanced hematologic malignancies, Phase II in pancreatic and other solid tumors).
All this demonstrates how promising new IMPDH inhibitors could be and why the inhibiting activity of compounds is worth being evaluated on such a highly pertinent target.

AVN-944	VX-148	VX-497
MPA (mycophenolic acid)	CellCept®	BMS-337197
Tiazofurin	Ribavirine	Mizoribine

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References (with links to PubMed) 1. L. Hedstrom and L. Gan (2006): IMP dehydrogenase: structural schizophrenia and an unusual base Curr. Opin. Chem. Biol. 10(5), 520-525 2. B. J. Barnes et al. (2001): Mechanism of action of the antitumor agents 6-benzoyl-3,3-disubstituted-1,5-diazabicyclo[3.1.0]hexane-2,4-diones: Potent inhibitors of human type II inosine 5'-monophosphate dehydrogenase Int. J. Cancer. 94(2), 275–281 3. R. E. Beevers et al. (2006): Low molecular weight indole fragments as IMPDH inhibitors Bioorg. Med. Chem. Lett. 16(9), 2535-2538 4. L. Chen and K. W. Pankiewicz (2007): Recent development of IMP dehydrogenase inhibitors for the treatment of cancer Curr. Opin. Drug Discov. Devel. 10(4):403-12 ( Review) 5. B. J. Barnes et al. (2001): Induction of Tmolt4 Leukemia Cell Death by 3,3-Disubstituted-6,6-pentamethylene-1,5-diazabicyclo[3.1.0]hexane-2,4-diones: Specificity for Type II Inosine 5'-Monophosphate Dehydrogenase J. Pharm. Exp. Therap. 298(2), 790-796 6. K. Malek et al. (2004): Effects of the IMP-dehydrogenase inhibitor, Tiazofurin, in bcr-abl positive acute myelogenous leukemia Leukemia Research 28, 1125–1136 7. L. Hong et al. (2006): ZNRD1 mediates resistance of gastric cancer cells to methotrexate by regulation of IMPDH2 and Bcl-2 Biochem. Cell Biol. 84(2): 199–206 8. S. Peñuelas et al. (2005): Modulation of IMPDH2, survivin, topoisomerase I and vimentin increases sensitivity to methotrexate in HT29 human colon cancer cells FEBS 272, 696–710 9. T. D. Colby et al. (1999): Crystal structure of human type II inosine monophosphate dehydrogenase: implications for ligand binding and drug design PNAS, 96(7), 3531–3536 10. E. J. Iwanowicz et al. (2003): Inhibitors of inosine monophosphate dehydrogenase: SARs about the N-[3-Methoxy-4-(5-oxazolyl)phenyl moiety Bioorg. Med. Chem. Lett. 13(12), 2059-2063 11. J. Jain et al. (2002): Characterization of pharmacological efficacy of VX-148, a new potent immunosuppressive inosine 5'-monophosphate dehydrogenase inhibitor J. Pharm. Exp. Therap. 302(3), 1272-1277 12. J. Jain et al. (2004): Regulation of inosine monophosphate dehydrogenase type I and type II isoforms in human lymphocytes Biochem. Pharmacol. 67(4), 767-776 13. G. M. Buckley et al. (2005): Quinazolinethiones and quinazolinediones, novel inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure–activity relationships Bioorg. Med. Chem. Lett. 15(3), 751-754 14. T. G. Murali Dhar et al. (2003): 3-Cyanoindole-Based Inhibitors of Inosine Monophosphate Dehydrogenase: Synthesis and Initial Structure–Activity Relationships Bioorg. Med. Chem. Lett. 13(20), 3557-3560