In this article, the projects in drug metabolism and pharmacokinetics funded by NSFC during "10th Five Year Plan" (2001~2005) in China were reviewed. The major research fields of these projects in drug metabolism and pharmacokinetics funded by NSFC during the financial years were overviewed and analyzed. Finally, current problems in these research fields in China were also briefly analyzed.

Identification and validation of a new target is one of the most important steps for new antituberculosis (TB) drug discovery. Researches have shown that Mycobacterium tuberculosis (Mtb) encodes 20 CYP450 enzymes which play important roles in the synthesis and metabolism of lipid, cholesterol utilization, and the electron transport of respiratory chain in Mtb. With the critical roles within the organism as well as the protein structures of six Mtb CYP450 enzymes being clarified, some of them have been highlighted as potential anti-tuberculosis targets. In this paper, the phylogenetic analysis, the structural features, and the enzymatic functions of Mtb CYPs, as well as the mechanism of interactions with selective inhibitors such as azole antifungal agents for the CYPs have been reviewed and summarized. The druggability of the CYPs has also been analyzed for their further utility as targets in high throughput screening and rational design of more selective inhibitors.

Aminoglycoside antibiotics, due to their strong antibacterial effects and broad antimicrobial spectra, have been very commonly used in clinical practice in the past half century. However, aminoglycoside antibiotics manifest severe ototoxicity and nephrotoxicity, and are one of top factors in hearing loss. In this study, three members of the aminoglycoside antibiotics family, gentamycin, neomycin and streptomycin, were chosen as the representatives to be investigated for their toxicity to the embryonic development and the larva hair cells in zebrafish, and also to their target genes associated with hearing-related genes. The results showed that: (1) the lethal effect of all three drugs demonstrated a significant dependence on concentration, and the severity order of the lethal effect was streptomycin > neomycin > gentamycin; (2) all the three drugs caused the larva trunk bending in resting state at 5 dpf (day past fertilization), probably due to their ototoxicity in the physical imbalance and postural abnormalities; (3) impairment and reducing of the hair cells were observed in all three cases of drug treatment; (4) four genes, eya1, val, otx2 and dlx6a, which play an important role in the development of hearing organs, showed differential and significant decrease of gene expression in a drug concentration-dependent manner. This study for the first time reports the relevance between the expression of hearing genes and the three ototoxic antibiotics and also proved the feasibility of establishing a simple, accurate, intuitive and fast model with zebrafish for the detection of drug ototoxicity.

Liver cancer is one of the most common neoplastic diseases with high mortality in China. Currently, antimicrotubule drugs such as paclitaxel (PTX) and vincristine (VCR), are used as the common agents in the clinical chemotherapy for liver cancer. However, the responses of patients to these drugs vary markedly. Successful identification of intracellular factors influencing liver cancer's sensitivity to antimicrotubule drugs would be of great clinical importance. In this study, by engineering human hepatoma cell HepG2 to overexpress synuclein-gamma (SNCG), we investigated if SNCG is a molecular factor associated with the sensitivity to antimicrotubule drug treatment. Real-time RT-PCR and Western blotting assays showed SNCG was successfully overexpressed in HepG2/ SNCG cells compared with HepG2/Neo cells. The overexpressed SNCG altered the proliferation activity in HepG2 cells, which was 66% higher than that of HepG2/Neo cells through MTT method. The overexpressed SNCG also reduced sensitivity of HepG2 cells to antimicrotubule drugs: after PTX or VCR treatment, the proportion of HepG2/SNCG cells in G2/M arrest was significantly lower than that in HepG2/Neo cells. Correspondingly, HepG2/SNCG cells showed significantly lower mitotic index than HepG2/Neo cells. Meanwhile, HepG2/SNCG cells showed higher resistance to PTX and VCR than HepG2/Neo cells, with resistance index 21 and 15 respectively. Our studies suggested that the overexpression of SNCG could confer resistance to antimicrotubule drugs in hepatoma cells; and it indicated that SNCG may be as a potential response marker for antimicrotubule drugs in liver cancer chemotherapy.

Chuangxinmycin (CM) from Actinoplanes tsinanensis was an antibiotic discovered by Chinese scientists about 40 years ago. It contains a new heterocyclic system of indole fused with dihydrothiopyran, whose biosynthetic mechanism remains unclear. CM is used as an oral medicine in the treatment of bacterial infections in China. The simple structure makes CM as an attractive candidate of structure modification for improvement of antibacterial activity. Recently, we analyzed the secondary metabolites of Actinoplanes tsinanensis CPCC 200056, a CM producing strain, as a natural CM analogue. We discovered the first natural CM analogue 3-demethylchuangxinmycin (DCM) as a new natural product. Compared to CM, DCM exhibited a much weaker activity in the inhibition of the bacterial strains tested. The finding provides valuable information for the structure-activity relationship in the biosynthesis of CM.

Objective To investigate the phenotypic and genetic characteristics of the lysostaphin‐resistant Staphylococcus aureus variants induced by recombinant lysostaphin in vitro .Methods Three clinical isolates of S . aureus ,including two resistant to methicillin (MRSA ) and one susceptible to methicillin (MSSA ) were induced by treatment with sub‐MIC of recombinant lysostaphin via one‐step selection in vitro .Susceptibility of the variants to antibiotics were determined and compared with their parental strains .The full length of femABX genes was amplified by polymerase chain reaction and sequenced to identify the potential mutation sites in these genes .The growth‐curve in liquid medium and virulence in a mouse systemic infection model of both parental and variant strains were observed . Results The frequency of lysostaphin resistance in S . aureus was between 10-4 to 10-8 following induction by lysostaphin . Resistance to lysostaphin was associated with a significant decrease in growth rate in vitro and virulence in vivo ,as well as increased susceptibility toβ‐lactams evidenced by the M IC of β‐lactams against the variants as low as 1/4 000 to 1/2 of the M IC against their parental strains . Sequencing of f emA BX genes showed mutation in femA gene in both variants ,which resulted in a premature termination codon .Conclusions Resistance of S . aureus to lysostaphin may develop following induction by recombinant lysostaphin in vitro . The lysostaphin‐resistant S . aureus variants are characteristic of lower growth rate , decreased virulence ,and higher susceptibility to β‐lactams .

Three hundred and twenty endophytic actinobacterial strains were isolated from psammophytes collected from Taklamakan Desert and identified.Among them,three strains already had been identified as new species of two genera and sixteen isolates showed relatively low 16S rRNA similarities＜98.6％to validly described species.Seventy-five of the isolates were selected as representative strains to screen antibacterial activity and mechanism.Forty-seven strains showed antagonistic activity against at least one of the indicator bacteria.Two Streptomyces strains produced bioactive compounds inducing DNA damage,and two Streptomyces strains produced bioactive compounds with inhibitory activity on protein biosynthesis.Notably,the strain Streptomyces sp.8P21H-1 that demonstrated both strong antibacterial activity and inhibitory activity on protein biosynthesis was prioritized for exploring new antibiotics.Under the strategy of integrating genetics-based discovery program and MS/MS-based molecular networking,two new streptogramin-type antibiotics,i.e.,acetyl-griseoviridin and desulphurizing gri-seoviridin,along with known griseoviridin,were isolated from the culture broth of strain 8P21H-1.Their chemical structures were determined by HR-MS,and 1D and 2D NMR.Desulphurizing griseoviridin and griseoviridin exhibited antibacterial activities by inhibiting translation.

The objective of this study was to investigate the genetic basis of high level aminoglycoside resistance in Acinetobacter baumannii clinical isolates from Beijing, China. 173 A. baumannii clinical isolates from hospitals in Beijing from 2006 to 2009 were first subjected to high level aminoglycoside resistance (HLAR, MIC to gentamicin and amikacin>512 µg/mL) phenotype selection by broth microdilution method. The strains were then subjected to genetic basis analysis by PCR detection of the aminoglycoside modifying enzyme genes (aac(3)-I, aac(3)-IIc, aac(6')-Ib, aac(6')-II, aph(4)-Ia, aph(3')-I, aph(3')-IIb, aph(3')-IIIa, aph(3')-VIa, aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id, ant(2″)-Ia, ant(3″)-I and ant(4')-Ia) and the 16S rRNA methylase genes (armA, rmtB and rmtC). Correlation analysis between the presence of aminoglycoside resistance gene and HLAR phenotype were performed by SPSS. Totally 102 (58.96%) HLAR isolates were selected. The HLAR rates for year 2006, 2007, 2008 and 2009 were 52.63%, 65.22%, 51.11% and 70.83%, respectively. Five modifying enzyme genes (aac(3)-I, detection rate of 65.69%; aac(6')-Ib, detection rate of 45.10%; aph(3')-I, detection rate of 47.06%; aph(3')-IIb, detection rate of 0.98%; ant(3″)-I, detection rate of 95.10%) and one methylase gene (armA, detection rate of 98.04%) were detected in the 102 A. baumannii with aac(3)-I+aac(6')-Ib+ant(3″)-I+armA (detection rate of 25.49%), aac(3)-I+aph(3')-I+ant(3″)-I+armA (detection rate of 21.57%) and ant(3″)-I+armA (detection rate of 12.75%) being the most prevalent gene profiles. The values of chi-square tests showed correlation of armA, ant(3″)-I, aac(3)-I, aph(3')-I and aac(6')-Ib with HLAR. armA had significant correlation (contingency coefficient 0.685) and good contingency with HLAR (kappa 0.940). The high rates of HLAR may cause a serious problem for combination therapy of aminoglycoside with β-lactams against A. baumannii infections. As armA was reported to be able to cause high level aminoglycoside resistance to most of the clinical important aminoglycosides (gentamicin, amikacin, tobramycin, etc), the function of aminoglycoside modifying enzyme gene(s) in A. baumannii carrying armA deserves further investigation.

(E)-Methyl-4-aryl-4-oxabut-2-enoate (YH-8) is a novel PKnB protein kinase inhibitor with good anti-tuberculosis activity. To evaluate its pharmacokinetics in rats, a sensitive and selective high performance liquid chromatography-tandem mass spectrometric (LC--MS/MS) method has been developed and validated for the quantification of YH-8 in rat plasma for the first time. Samples were pre-treated using a liquid--liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column by gradient elution with methanol--water as the mobile phase. YH-8 was detected using a tandem mass spectrometer in positive selected reaction monitoring (SRM) mode. Method validation revealed good linearity over the range of 1-500 ng/mL for YH-8 with a lower limit of quantification (LLOQ) of 1 ng/mL. Intra- and inter-day precision of YH-8 assay in rat plasma samples were 2.0%-6.8%, with accuracy of the method being 100.69%-106.18%. Stability test showed that when spiked into rat plasma, YH-8 was stable for 12 h at room temperature, for up to 15 days at -70 °C, and after three freeze-thaw cycles. Extracted samples were found to be stable over 12 h in an auto-sampler. The method was successfully applied to the pharmacokinetic study of YH-8 in rats after oral administration at 100 mg/kg and 200 mg/kg.

Bacteremia is a life-threating syndrome often caused by methicillin-resistant (MRSA). Thus, there is an urgent need to develop novel approaches to successfully treat this infection. Staphylococcal accessory regulator A (SarA), a global virulence regulator, plays a critical role in pathogenesis and -lactam antibiotic resistance in . Hypericin is believed to act as an antibiotic, antidepressant, antiviral and non-specific kinase inhibitor. In the current study, we investigated the impact of hypericin on -lactam antibiotics susceptibility and mechanism(s) of its activity. We demonstrated that hypericin significantly decreased the minimum inhibitory concentrations of -lactam antibiotics (.., oxacillin, cefazolin and nafcillin), biofilm formation and fibronectin binding in MRSA strain JE2. In addition, hypericin significantly reduced expression, and subsequently decreased and virulence-related regulators (.., ) and genes (.., and ) expression in the studied MRSA strain. Importantly, the synergistic effect of hypericin with -lactam antibiotic (.., oxacillin) translated into therapeutic outcome in a murine MRSA bacteremia model. These findings suggest that hypericin plays an important role in abrogation of -lactam resistance against MRSA through inhibition, and may allow us to repurpose the use of -lactam antibiotics, which are normally ineffective in the treatment of MRSA infections (.., oxacillin).