Structure guided development of novel thymidine mimetics targeting Pseudomonas aeruginosa thymidylate kinase: from hit to lead generation.

Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of an essential component, thymidine triphosphate, in DNA replication. All reported TMK inhibitors are thymidine analogues, which might retard their development as potent therapeutics due to cell permeability and off-target activity against human TMK. A small molecule hit (1, IC(50) = 58 µM), which has reasonable inhibition potency against Pseudomonas aeruginosa TMK (PaTMK), was identified by the analysis of the binding mode of thymidine or TP(5)A in a PaTMK homology model. This hit (1) was cocrystallized with PaTMK, and several potent PaTMK inhibitors (leads, 46, 47, 48, and 56, IC(50) = 100-200 nM) were synthesized using computer-aided design approaches including virtual synthesis/screening, which was used to guide the design of inhibitors. The binding mode of the optimized leads in PaTMK overlaps with that of other bacterial TMKs but not with human TMK, which shares few common features with the bacterial enzymes. Therefore, the optimized TMK inhibitors described here should be useful for the development of antibacterial agents targeting TMK without undesired off-target effects. In addition, an inhibition mechanism associated with the LID loop, which mimics the process of phosphate transfer from ATP to dTMP, was proposed based on X-ray cocrystal structures, homology models, and structure-activity relationship results.

Purification of the large ribosomal subunit via its association with the small subunit.

We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.

Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway.

The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes. Inhibition of ATP consumed during the MurC reaction, using an adaptation of a kinase assay format, identified a number of potential inhibitory chemotypes. After nonspecific inhibition testing and chemical attractiveness were assessed, C-1 emerged as a compound for further characterization. The inhibition of MurC by this compound was confirmed in both a kinetic-coupled enzyme assay and a direct nuclear magnetic resonance product detection assay. C-1 was found to be a low micromolar inhibitor of the E. coli MurC reaction, with preferential inhibition by one of two enantiomeric forms. Experiments indicated that it was a competitive inhibitor of ATP binding to the MurC enzyme. Further work with MurC enzymes from several bacterial sources revealed that while the compound was equally effective at inhibiting MurC from genera (Proteus mirabilis and Klebsiella pneumoniae) closely related to E. coli, MurC enzymes from more distant Gram-negative species such as Haemophilus influenzae, Acinetobacter baylyi, and Pseudomonas aeruginosa were not inhibited.