ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS. ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics.

We rationalize the behaviour of protonated merocyanines in water through cross-validation of 1H NMR, UV-Vis and pH measurements, and show their capability to act as reversible photoacids along light/dark cycles can be described by a four-state model.

Positron emission tomography (PET) is used in drug development to assist dose selection and to establish the relationship between blood and tissue pharmacokinetics (PKs). We present a new biomathematical approach that allows prediction of repeat-dose (RD) brain target occupancy (TO) using occupancy data obtained after administration of a single dose (SD). A PET study incorporating a sequential adaptive design was conducted in 10 healthy male adults who underwent 4 PET scans with [(11)C]DASB ([(11)C]N,N-dimethyl-2-(2-amino-4-cyanophenylthio) benzylamine): 1 at baseline, 2 after 20 mg SD of the 5-hydroxytryptamine transporter (5-HTT) inhibitor duloxetine, and 1 after 4 days daily administration of 20 mg duloxetine. An adaptive design was used to select optimal times after SD for measurement of occupancy. Both direct and indirect PK/TO models were fitted to the SD data to characterise the model parameters and then applied to a predicted RD duloxetine plasma time course to predict the 5-HTT occupancy after RD. Repeat-dose prediction from the indirect model (OC(50)=2.62±0.93 ng/mL) was significantly better (P<0.05) than that from the direct model (OC(50)=2.29±1.11 ng/mL). This approach increases the value of SD occupancy studies that are performed as part of first time in human drug development programmes by providing an estimate of the dose required to achieve the desired TO at RD.

The data demonstrate the sleep-promoting properties of the orexin antagonist SB-649868 in male patients with insomnia.

CTP binding and hydrolysis concentrate ParB at parS centromeres to mediate faithful chromosome segregation in bacteria.

The current interest in developing Glycine transporter Type 1 (GlyT-1) inhibitors, for diseases such as schizophrenia, has led to the demand for a GlyT-1 PET molecular imaging tool to aid drug development and dose selection. We report on [(11) C]GSK931145 as a novel GlyT-1 imaging probe in primate and man. Primate PET studies were performed to determine the level of specific binding following homologous competition with GSK931145 and the plasma-occupancy relationship of the GlyT-1 inhibitor GSK1018921. Human PET studies were performed to determine the test-retest reproducibility of [(11) C]GSK931145 and the plasma-occupancy relationship of GSK1018921. [(11) C]GSK931145 entered primate and human brain and yielded a heterogeneous pattern of uptake which was similar in both species with highest uptake in midbrain, thalamus, and cerebellum. Homologous competition in primates indicated no viable reference region and gave binding potential estimates between 1.5 and 3 for midbrain, thalamus and cerebellum, While the distribution and binding potential values were similar across species, both the plasma free fraction (f(P) : 0.8 vs. 8%) and delivery (K(1) : 0.025 vs. 0.126 ml cm(-3) min(-1) ) were significantly lower in humans. Test-retest reproducibility in humans calculated using a two tissue compartmental model was poor (VAR(V(T) ): 29-38%), but was improved using a pseudo reference tissue model (VAR(BP(ND) ): 16-23%). GSK1018921 EC(50) estimates were 22.5 and 45.7 ng/ml in primates and humans, respectively.

These results indicate a relationship between PK, target engagement and PD, suggesting a selective inhibition of recruitment of CCR6 cells by GSK3050002 and support further development of GSK3050002 in autoimmune and inflammatory diseases.

The orexin system plays a major role in the integration of metabolic and circadian influences that drive wakefulness. This paper describes initial Phase I trials of a novel dual orexin receptor antagonist SB-649868 that has demonstrated preclinical potential for treatment of sleep disorders. The trial designs included a single ascending dose escalation study (dose range: 10-80 mg in the fed and fasted states) and a multiple repeat dose study (dose range: 5-30 mg in the fed state) enrolling a total of 103 male volunteer subjects. SB-649868 was well tolerated at all doses in this study population, with mechanism-related adverse events (e.g. somnolence and fatigue) observed in a majority of subjects after 60 and 80 mg single doses. Although total drug exposure was similar in the fed and fasted states, the rate, but not the extent, of absorption increased in the fed state, resulting in an increased C(max). The typical estimated half-life of SB-649868 was 3-6 h - comparable with currently used hypnotic agents. Repeated administration of SB-649868 dose-dependently increased exposure to simvastatin (10 mg), suggesting CYP3A4 inhibition ranging from very mild (5 mg) to strong (30 mg). Evening dosing resulted in significant dose-dependent improvement in latency to persistent sleep, total sleep time and wake after sleep onset as measured by polysomnography. Next-morning testing did not detect evidence of residual cognitive effects. Results of these trials support further investigation of SB-649868 and other dual orexin receptor antagonists as potentially effective and well-tolerated treatments for patients with sleep disorders.