Amidotransferase inhibitors

Figure 4. Schematic representation ofAcivicin s mode of action as well as that of other amidotransferase inhibitors, where X is the pharmacophoric moiety of cysteine protease inhibitors.

Table 7. Fungicidal Activity of Amidotransferase Inhibitors Derived from P-Amino-alanine.

Indirect Pathway Inhibitors of Glutamyl-tRNA Synthetase, Aspartyl-tRNA Synthetase, and Aminoacyl-tRNA Amidotransferase... 

Table 5 Inhibitors of Helicobacter pylori GatCAB amidotransferase derived from puromycin...

GatCAB amidotransferase.This natural product mimics the charged 3 -terminus of aa-tRNA and has been used as a tool for the study of protein biosynthesis. The parent compound 22 is a very weak inhibitor of AdT. The amino acid chain is related to tyrosine and differs from the glutamic and aspartic side chains transformed in the kinase or the transamidase steps. Replacement of the methoxyphenyl moiety of puromycin by carboxylic acid derivatives (23-26) improved the ability to inhibit this AdT. Stable analogues of the transition state in the last step of the transamidation process (27-29) where the carbonyl to be attacked by NH3 is replaced by tetrahedral sulfur or phosphorus atom with a methyl group mimicking ammonia exhibited the highest activity. 

Robert Chenevert is Professor of Organic Chemistry at Universite Laval, Quebec, Canada. He studied chemistry (B.Sc. and M.Sc.) at the Universite de Montreal. After receiving his Ph.D. in organic chemistry in 1975 at the Universite de Sherbrooke under the supervision of Professor Pierre Deslongchamps, he spent a postdoctoral year at Harvard (R. B. Woodward s group). His main research interest is the application of biocatalysts in asymmetric synthesis. He is also interested in the design of inhibitors of enzymes involved in the aminoacylation of tRNA (aminoacyl-tRNA synthetases and aminoacyl-tRNA amidotransferases). 

In addition to the analogues listed in Table 2.3, cordycepin [302]. 3 -amino-3 -deoxyadenosine [173], and formycin [303] can inhibit the de novo pathway by blocking the phosphoribosylpyrophosphate amidotransferase. Thus, a number ofpurine analogues—after anabolism to nucleoside phosphates—can act as feedback inhibitors, and this inhibition may be the primary cause of their cytotoxicity. 

FIGURE 22-48 Azaserine and acivicin, inhibitors of glutamine amidotransferases. These analogs of glutamine interfere in a number of amino acid and nucleotide biosynthetic pathways. 

Inhibitors of Nucleotide Biosynthesis Suggest mechanisms for the inhibition of (a) alanine racemase by l-fluoroalanine and (b) glutamine amidotransferases by aza-serine. 

The diazo compound 0-(2-diazoacetyl)-L-serine, known also as azaserine (see Fig. 22-48), is a powerful inhibitor of glutamine amidotransferases. If growing cells are treated with azaserine, what intermediates of nucleotide biosynthesis would accumulate Explain. 

Acivicin is a potent inhibitor of several steps in purine nucleotide biosynthesis that utilize glutamine. The enzymes it inhibits are glutamine PRPP amidotransferase (step 1, fig. 23.10), phosphoribosyl-A-formylglycinamidine synthase (step 4, fig. 23.10), and GMP synthase (see fig. 23.11). In pyrimidine nucleotide biosynthesis the enzymes inhibited are carbamoyl synthase (step 1, fig. 23.13) and CTP synthase (see fig. 23.14). Acivicin is under trial for the treatment of some forms of cancer. 

Acivicin is a cjhostatic antibiotic, a glutamine analogue, which is a potent inhibitor of 1-asparagine synthetase and other 1-glutamine amidotransferases and has its cjhotoxic action by blocking nucleotide biosynthesis. 

Azaserine is an antibiotic that is a potent inhibitor of purine nucleotide synthesis. Azaserine is similar in structure to glutamine and is an irreversible inhibitor of glutamine amidotransferases, which catalyze the ATP-dependent transfer of the amido nitrogen of glutamine to an acceptor. Four such reactions occur in nucleotide synthesis. 

GMP synthesis The two reactions of GMP synthesis are an NAD -dependent oxidation followed by an amidotransferase reaction. In Step 1, IMP dehydrogenase employs the substrates NAD and H2O in catalyzing oxidation of IMP at C-2. The products are xanthylic acid (XMP or xanthosine monophosphate), NADH, and H. GMP is a competitive inhibitor (with respect to IMP) of IMP dehydrogenase. In Step 2, transfer of the amido-N of glutamine to the C-2 position of XMP yields GMP. This ATP-dependent reaction is catalyzed by GMP synthetase. 

Several other inhibitors of amidotransferases are known (24,25), and several are also known to have fungicidal activity (28). They fit into the scheme shown above, where X is part of a cysteine protease inhibitor (29). 

The ratio of the Michaelis constant of glutamine to inhibitor constants for azaserine and diazo-oxo-norleucine for the two amidotransferases of purine biosynthesis in pigeon liver are given in the accompanying tabulation. 

Early in vivo studies with bacterial suspensions indicated that purines exerted a profound inhibitory effect on an early enzyme of the pathway [39-41]. The nature of the inhibitor could not be readily determined because of the interconversion reactions in the whole-cell preparations. The use of special mutants deficient in certain interconversion steps implicated an adenine derivative as the effector in one case [42] and a guanine derivative in another [33]. Axiomatic inference suggested that the feedback-inhibited enzyme should be the first unique enzyme in the pathway, the glutamine-dependent PRPP amidotransferase (EC 2.4.4. 

Effect of actinomyin JD, cycloheximide, and azacytidine on de novo purine biosynthesis Table 2 illustrates that all three inhibitors, actinomycin D, cycloheximide, and azacytidine inhibit de novo purine biosynthesis as measured by the incorporation of -formate into total purines Labelling was for 2 hours, and the results are the average of three experiments Controls had between 25,000 and 30,000 cpm/10 cells Actinomycin D had no effect on Li vitro assayed PRPP amidotransferase or PRPP synthetase activities for cell pretreated for 2 hours with the drug ... 

Increases in rates of purine production could in theory result from increased concentrations of the substrates PP-ribose-P or glutamine, increased activity of the amidotransferase, or decreased availability or non-optimal ratios of feedback inhibitors [39,46]. 

If the human enzyme is not saturated with glutamine, variations in glutamine concentrations would be expected to affect the rate of purine biosynthesis, although less exquisitely than variations in concentrations of PP-ribose-P. The kinetics of the amidotransferase reaction are hyperbolic with respect to glutamine at all levels of PP-ribose-P, in the presence or absence of nucleotide inhibitors [47,63]. Purine ribonucleotide inhibition is non-competitive with respect to glutamine [47,63] and glutamine does not reverse the association of amidotransferase subunits caused by ribonucleotides [48]. 

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