Cytidine deaminase inhibitors

Susceptibility to carboxylesterase in human liver cell culture with cytidine deaminase inhibitor tetrahydrouridine (mmol/mg protein/h). 

It should therefore be possible to design chemical structures, modeled after known or putative reaction intermediates that resemble postulated transition states. They may exhibit high affinities for the reactive sites of enzymes and therefore function as effective, but reversible, inhibitors. A successful example is the potent and specific cytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine, which effectively blocks the conversion of cytidine to uridine. It was similarly demonstrated that 1,6-dihydro-6-hydroxymethylpurine effectively blocked the deamination of adenine to hypoxanthine by adenine deaminase (Eq. 2.13). 

M. D. Erion and M. R. Reddy, J. Am. Chem. Soc., 120,3295 (1998). Calculation of Relative Hydration Free Energy Differences for Heteroaromatic Compounds Use in the Design of A nosine Deaminase and Cytidine Deaminase Inhibitors. 

The susceptibilities of some of these fluorinated purine nucleosides to the action of enzymes are now described. In contrast to the inertness of the 2 -deoxy-2 -fluoro- and 3 -deoxy-3 -fluorocytidine analogs 739, 744, and 821 towards cytidine deaminase, the adenosine compounds 867, 883, and 906 are readily deaminated - by the adenosine deaminase in erythrocytes and calf intestine, but the resulting (deaminated) inosine compounds (from 867 and 883), as well as 888, are highly resistant - to cleavage by purine nucleoside phosphorylase (to give hypoxanthine base for the first two). The reason was discussed. Both 867 and 883 can form the 5 -triphosphates, without deamination, in human erythrocytes or murine sarcoma cells in the presence of 2 -deoxycoformycin, an adenosine deaminase inhibitor, but... 

The potency of zebularine is about 10-fold lower than for the azacytosines [73]. Zebularine also inhibits cytidine deaminase [75] which is involved in nucleoside catabolism and deactivates also for example azacitidine and its desoxy analog [76]. Thus, it increases the concentrations of nucleoside triphosphates for incorporation into DNA, the efficacy of DNA methylation and ultimately the anticancer activity of for example azacitidine [77, 78[. Zebularine is metabolized by aldehyde oxidase and ithasbeen shovm that its activity can be increases if an inhibitor of that enzyme, for example raloxifene is given in combination [79]. One big question about all epigenetic drugs is the origin of the observed selectivity towards cancer cells. For zebularine, it has been shown that much less activation towards triphosphate metabolites that can be incorporated into DNA occurs in normal muscle tissue as compared to cancer tissue [80]. 

Laliberte, J., Marquez, V.E. and Momparler, R.L. (1992) Potent inhibitors for the deamination of cytosine arabinoside and 5-aza-2 -deoxycytidine by human cytidine deaminase. 

Liu and co-workers treated the (V,N -bis(trimethylsilyl) derivatives of 104 with 2,3,5-tris-O-benzoyl-D-ribosyl bromide. Debenzoylation of the intermediate product afforded /J-D-ribofuranosyl-/V,/V -pentameth-yleneurea nucleoside 137, found to be a cytidine deaminase (CDA) inhibitor against mouse kidney enzyme (although less effective than the seven- or six-membered ring homologs) (81JMC662). 

Transition-state analogs are potent inhibitors of enzymes. In the enzyme cytidine deaminase from the bacterium E. coli, the following chemical transformation takes place ... 

Flucytosine is a powerful antifungal agent used in the treatment of serious systemic fungal infections, such as Cryptococcus neoformans and Candida spp (Table 40.2). Flucytosine itself is not cytotoxic but, rather, is a pro-drug that is taken up by fungi and metabolized to 5-fluorouracil (5-FU) by fungal cytidine deaminase (Fig. 40.11) (51). Then, 5-FU is converted to 5-fluorodeoxyuridine, which as a thymidylate synthase inhibitor interferes with both protein and RNA biosynthesis. 5-Fluorouracil is cytotoxic and is employed in cancer chemotherapy (see Chapter 42). Human cells do not contain cytosine deaminase and, therefore, do not convert flucytosine to 5-FU. Some intestinal flora, however, do convert the drug to 5-FU, so human toxicity does result from this metabolism. Resistance rapidly develops to flucytosine when used alone, so it is almost always used in conjunction with amphotericin B. Use of flucytosine has declined since the discovery of fluconazole. 

Similarly, 3,4,5,6-tetrahydrouridine 9.76) is a potent and specific inhibitor for the deamination of cytidine 9.75) by cytidine deaminase (Carmenier, 1968). The tight binding of this inhibitor is attributed to its resemblance, in the critical position, to a tetrahedral intermediate analogous to 9.73). Similarly, adenosine deaminase is strongly inhibited by l,6>dihydro-6-hydroxymethylpurine 9.77) (Evans and Wolfenden, 1970). 

Conventional methods have been used to prepare ribonucleoside analogues from 2-(l//)pyrazinone, 3,5-disubstituted pyridines (including a-anomers), 6-methyl-l,3-oxazine-2,4-dione and 6-methyluracil, 2-alkyl-4,5-dicarboxamido-imidazoles, 7-membered ring analogues (4) and (5) of uracil and tetrahydro-uracil [one diastereomer of (4) being the most potent inhibitor of cytidine deaminase yet discovered], 2-alkylthioadenine, 8-aza-3-deaza-guanine/ ... 

The chart in Fig. 2 shows an alternate path for the formation of dUMP by direct deamination of dCMP. This may be how cytidine could be converted to thymidylate in the cases cited above [125,126]. However, this deaminase is not usually detected in E. coli but is induced by infection with T(even) phages [132,133]. It has also been purified from chick embryo and mammalian tissues, and its properties have been extensively analyzed [134-136]. It acts as a typical allosteric enzyme in both the phage-infected E. coli and animal systems. Homotropic substrate interaction is evident, and this is modified by dCTP as an activator, and by dTTP (sometimes dGMP) as an allosteric inhibitor. This type of control apparently functions to regulate the level of dTTP by feedback inhibition and by activation when the supply of dTTP is depleted. Cytidine deaminase (EC 3.5.4.5) isolated from sheep liver [137] appears to have the same allosteric properties, with the same positive and negative effectors, as those of dCMP deaminase. The latter enzyme is also induced by phage infection in B. subtiUs, and in contrast to the deaminase from all other sources it does not show allosteric inhibition or activation by any nucleotide [138]. 

We treated a PNP-deficient patient, born in January 1975, (8), with erythrocyte transfusions for more than 5 years. This regimen has resulted in a partial restoration of in vitro T cell function. Orally, and lateron intravenously administered dCyd had no additional benificial effect at all(6). The still existing T cell deficiency is reflected in the clinical condition of the patient she still shows a vulnerability to infection(9). We therefore have treated the patient again with dCyd, this time in combination with tetrahydro-uridine (THU), a potent non-toxic inhibitor of (deoxy)cytidine deaminase. We present here the metabolic and immunological results during this treatment. 

The in vitro cytotoxicity of DMDC against L1210 cells was prevented dose depen-dently by 2 -deoxycytidine, suggesting that DMDC is phosphorylated by deoxycytidine kinase. Unlike ara-C, DMDC was not a substrate of cytidine deaminase from mouse kidney.33 Together with these characteristics, chemically synthesized DMDCTP was a potent inhibitor of DNA polymerase a, P, and y from calf thymus with Ki values of 0.42, 2.52, and 1.00 pM, respectively, in a manner competitive with dCTP, while ara-CTP inhibited only DNA polymerase a with a K of 1.10 pM.32 it was also found that DMDCTP was incorporated into DNA molecules at the site complementary to guanine by the action of DNA... 

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