## 2 Results
### Amino Acids, Amino Acid Derivatives and Nucleotides Show Major Differences after Infection
#### 2.4.1 Inhibition of Purine and Pyrimidine Synthesis by DON and Azaserine  
Since both inhibitors are reported to vary in their target enzymes and efficiency [27; 28], we performed the infection experiment and added DON or azaserine in growth inhibitory concentrations afterwards to separate cultures. Due to the inhibition we expected educts of glutamine transferase reactions in increased concentrations. Indeed, the amount of PRPP, the educt of the glutamine transaminase reaction by the phosphoribosylpyrophosphate amidotransferase (PPAT), was increased 17 fold in uninfected A549 cells in the presence of DON (Figure 5A). The same is true for 5\({}^{\prime}\)-phosphoribosyl-\(N\)-formylglycinamide (FGAR) the educt of the transaminase reaction by the phosphoribosylformylglycinamidine synthase (PFAS) when cells were treated with DON (14 fold) or azaserine (300 fold) (Figure 5A). The accumulation of FGAR in DON treated cells was probably lower due to the additional upstream inhibition of the PPAT. Whereas PRPP was not increased in infected cells treated with the inhibitor, the FGAR level was also elevated eight fold in infected cells treated with azaserine. However, it was still only slightly above the detection limit. The next glutamine dependent reaction in purine synthesis is the conversion of XMP to GMP by the GMP-synthetase. XMP was solely detected in DON treated cells indicating an inhibition of this enzyme in the presence of DON but again not azaserine (Figure 5B). Despite the described potential inhibition sites in the purine synthesis under control conditions, ATP and GTP were still present in the cell but in 1.6- and 2.6-fold decreased amounts. Noteworthy, GMP, IMP and AMP amounts increased by 13, 30 and four fold, respectively, in infected cell cultures treated with azaserine. This indicates that azaserine only affects glutamine dependent reactions, but also might exhibit secondary effects that appear only under infection conditions (Figure 5B). A glutamine using step in the synthesis of pyrimidines is the ligase reaction performed by the CTP-synthetase. A549 cells treated with DON showed strongly decreased amounts (12-fold lower) of the nucleotide CTP, which is an abundant metabolite under control and infection conditions (Figure 5C). Consequently, we found that CTP dependent metabolites also decreased in concentration. We detected CDP, the DNA precursor 2-deoxy-CTP and a proteoglycan precursor CMP-neuraminate in 13-fold, 18-fold and nine-fold lower amounts, respectively. Additionally, DON treated cells showed two- to three-fold higher amounts of UTP which is the direct precursor of CTP. Similar effects were observed for UTP derived metabolites such as UDP, UDP-Glc, UDP-GlcA and UDP-pentose. Azaserine on the other hand showed no inhibitory effect on CTP synthesis since a two- to three-fold increase in CTP and CDP concentration under control conditions was measured in treated cells. Interestingly, infected cells treated with either DON or azaserine showed no decline in CTP concentration but an increase up to 3.6 fold. These data show that: (i) azaserine has less target enzymes than DON in A549 cells at the chosen concentration; and (ii) infected cells are either less susceptible to inhibitor treatment, or the reactions that are supposed to be inhibited are not active under infection conditions. Figure 5: Inhibition of nucleotide synthesis with glutamine analogues. Control cells and infected cells were treated with either DON or azaserine (AZA) for 12 h. Identified educts and products of glutamine dependent reactions involved in purine and pyrimidine synthesis are displayed in relative concentrations as mean with standard deviation normalized on 10\({}^{7}\) cells (\(n\geq\) 7 out of at least four independent experiments).