, 2007; Meier et al., 2008; Pereira et al., 2009). In this study, we evaluate the inhibitory activity of PYRH-1 (sodium 3-[4-tert-butyl-3-(9H-xanthen-9-ylacetylamino)phenyl]-1-cyclohexylmethylpropoxycarbonyloxyacetate)

as a potential antimicrobial agent by targeting the bacterial UMP kinase, PyrH, which serves as a kinase in de novo pyrimidine biosynthesis pathway required for the growth of certain bacteria such as S. pneumoniae (Thanassi et al., 2002; Song et al., 2005) and H. influenzae (Akerley et al., 2002). PYRH-1 was discovered in the course of a 1536-well high throughput screening of an in-house large chemical library by the selection of chemicals directly inhibiting PyrH of S. pneumoniae. To test the inhibitory activity of PYRH-1 against PyrH, we used a luminescence-based ATP quantitative reagent. Moreover, molecular interaction analysis between PYRH-1 and S. pneumoniae CT99021 PyrH by surface plasmon resonance (SPR) and susceptibility tests of PYRH-1 against some bacteria were Akt inhibitor performed. This is the first report that PYRH-1 inhibits PyrH. Bacterial strains used in this study are described in Table 1. Escherichia coli DH5α (competent high E. coli DH5α, Toyobo Co., Ltd.) was used for the cloning of PyrH. Escherichia

coli Rosetta-Gami B (DE3) (Novagen) was used as the host for recombinant protein expression. These were grown at 35 °C in Luria–Bertani (LB) broth or LB agar (BD Biosciences) containing 100 μg mL−1 of carbenicillin (Sigma). The culture medium used Baricitinib for each bacterium is as follows: S. pneumoniae, cation-adjusted Mueller–Hinton Broth (CAMHB; BD Biosciences) containing 5% of lysed horse blood (Nippon Bio-Test Laboratories Inc.) or Todd Hewitt Broth (Becton, Dickinson and Co.); S. aureus and E. coli, CAMHB; H. influenzae, Haemophilus test medium [CAMHB containing 5 mg mL−1 of Yeast Extract (BD Biosciences), 15 μg mL−1 of Hemin (Sigma) and 15 μg mL−1 of β-NAD (Sigma)]. This strain was constructed by deleting

the acrA gene and replacing it with a gene that confers resistance to chloramphenicol (cat) Streptococcus pneumoniae TIGR4 and H. influenzae Rd KW20 genomic DNA were extracted with a DNeasy Tissue Kit (Qiagen). Plasmid DNA was extracted with a QIAprep Spin Miniprep Kit (Qiagen). PCR products and plasmids digested by restriction enzyme were purified with a QIAquick PCR Purification Kit (Qiagen). PCR products digested by restriction enzyme were purified with a MinElute Reaction Cleanup Kit (Qiagen). The open reading frame of the pyrH gene was amplified from S. pneumoniae TIGR4 genomic DNA with primers SpPyrH-N-XhoI (5′- CCG CTC GAG GTG AAA ATG GCG AAT CCC AAG T -3′) and SpPyrH-C-BamHI (5′- CGC GGA TCC TTA TTC CTT TTC TTC GAT ATT ATT TGA AAC TGT TG -3′). The open reading frame of pyrH was amplified from H.