The crude product was diluted with ethyl acetate (30 mL) and washed with saturated aq. TMK without undesired off-target effects. In addition, an inhibition mechanism associated with the LID loop, which mimics the (R)-Oxiracetam process of phosphate transfer from ATP to dTMP, was proposed based on X-ray co-crystal structures, homology models, and SAR results. INTRODUCTION Most classes of antibacterial agents currently employed in clinical use were discovered over 45 years ago, with the exception of the oxazolidinone class (Linezolid), introduced in 2000 to treat Gram positive infections. New (R)-Oxiracetam antibacterial therapeutics which utilize new mechanisms of action are urgently needed to combat growing resistance to existing antibacterial agents for both Gram positive and Gram negative infections. Although discovery of new antibacterial classes is extraordinarily difficult,1 the need is especially high for Gram negative organisms prevalent in the hospital and in particular for infections caused by (Pa), for which treatment options are often limited.2C4 Thymidylate kinase (TMK) has emerged as an attractive therapeutic target because inhibiting TMK functions blocks DNA synthesis in replicating organisms.5 TMK phosphorylates thymidine monophosphate (dTMP) to thymidine diphosphate (dTDP), using ATP as a phosphoryl donor.6 In addition, TMK is the last specific enzyme in the pathways for the synthesis of thymidine triphosphate (dTTP), which is an essential component in DNA synthesis.7 Therefore, targeting bacterial TMK has been the subject of recent investigation with inhibitors of TMK22 with the thymidine based dual substrate inhibitor TP5A led us to intimately understand the interactions required for thymidine binding to its binding site of PaTMK (Figure 2(a)). Furthermore, we found that a commercial compound, 1-methyl-6-phenyl imidazopyridinone (1), has similar chemical properties to thymidine, although their two-dimensional structures have low similarity. Flexible alignment of 1 1 to thymidine structure was performed to recognize their geometrical similarity using Molecular Operating Environment program (MOE).23 The result showed that 1 and thymidine are well overlapped with identical pharmacophore (Figure 2(b)). In the enzyme assay, compound 1 proved to be an inhibitor of PaTMK with approximately three-fold less potency than the known TMK inhibitor, dFTM (IC50 = 58 M vs. 20 M in Table 1). Open in a separate window Figure 2 Key interactions of dTMP and 1 in PaTMK. (a) Thymidine interactions in the active site of PaTMK at the homology model are represented in two-dimensional view; (b) The result of flexible alignment of 1 1 to dTMP shows clear pharmacophore match between 1 and dTMP. The structure of dTMP is represented as red line, and that of 1 1 is as stick with atom-type color (gray for carbon, blue for nitrogen, red for oxygen, and orange for phosphorous); (c) The overall X-ray co-crystal structure of PaTMK and 1 shows 1 binds (R)-Oxiracetam where dTMP bound (left panel). The structure of TP5A was merged from the reported X-ray structure (PDB ID: 4TMK) to compare similarity of binding site of (R)-Oxiracetam 1 1 and TP5A. The binding mode of 1 1 in PaTMK at the X-ray co-crystal structure shows the binding pose of 1 1 at PaTMK is identical to that of dTMP. -Helix is red, -sheet is yellow, and loop is green (left panel). The water molecule is represented with a Mmp17 red sphere, 1 and amino acids associated with the binding of 1 1 are represented as sticks with atom-type color, and H-bond interactions are represented with black lines. -Cation stacking between the phenyl ring of 1 1 and Arg96 is not marked. Hydrogen atoms are omitted for clarity. Figures are generated (R)-Oxiracetam with Pymol program. Table 1 PA01 and PAO280 (MexABoprM, MexXY, MexZ efflux pump knock out) and were found to have MICs 256 g/mL. Inhibitors 46 (and.