Discovery of TAK-960: An orally available small molecule inhibitor of polo-like kinase 1 (PLK1)

Using structure-based drug design, we identified and optimized a novel series of pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced solid malignancies.     

Nonbenzamidine acylsulfonamide tissue factor–factor VIIa inhibitors

Aminoisoquinoline and isoquinoline groups have successfully replaced the more basic P1 benzamidine group of an acylsulfonamide factor VIIa inhibitor. Inhibitory activity was optimized by the identification of additional hydrophobic and hydrophilic P′ binding interactions. The molecular details of these interactions were elucidated by X-ray crystallography and molecular modeling. We also show that decreasing the basicity of the P1 group results in improved oral bioavailability in this chemotype.     

Synthesis and acrosin inhibitory activities of 5-phenyl-1H-pyrazole-3-carboxylic acid amide derivatives

A series of novel 5-phenyl-1H-pyrazole-3-carboxylic acid amide derivatives were designed, synthesized, and their acrosin inhibitory activities in vitro were evaluated. The results of the acrosin inhibitory activity showed that all target compounds were more potent than control TLCK. Compounds AQ-A1, AQ-D3, AQ-D4, AQ-E4 and AQ-E5 exhibited stronger acrosin inhibitory activities than control ISO-1. Especially, compound AQ-E5 displayed the most potent acrosin inhibitory activity in all the compounds, with an IC₅₀ of 0.01 μmol/mL. This study provided a new structural class for the development of novel acrosin inhibitory agents.     

Discovery of an intravenous hepatoselective glucokinase activator for the treatment of inpatient hyperglycemia

Glucokinase (hexokinase IV) continues to be a compelling target for the treatment of type 2 diabetes given the wealth of supporting human genetics data and numerous reports of robust clinical glucose lowering in patients treated with small molecule allosteric activators. Recent work has demonstrated the ability of hepatoselective activators to deliver glucose lowering efficacy with minimal risk of hypoglycemia. While orally administered agents require a considerable degree of passive permeability to promote suitable exposures, there is no such restriction on intravenously delivered drugs. Therefore, minimization of membrane diffusion in the context of an intravenously agent should ensure optimal hepatic targeting and therapeutic index. This work details the identification a hepatoselective GKA exhibiting the aforementioned properties.     

Genetic diversity of Bartonella quintana in macaques suggests zoonotic origin of trench fever

Bartonella quintana is a bacterium that causes a broad spectrum of diseases in humans including trench fever. Humans were previously considered to be the primary, if not the only, reservoir hosts for B. quintana. To identify the animal reservoir and extend our understanding of the ecological and evolutionary history of B. quintana, we examined blood samples from macaques and performed multilocus sequence typing (MLST) analysis. We demonstrated the prevalence of B. quintana infection was common in macaques from main primate centres in mainland China. Overall, 18.0% (59/328) of rhesus macaques and 12.7% (39/308) of cynomolgus macaques were found to be infected with B. quintana by blood culture and/or polymerase chain reaction. The infection was more frequently identified in juvenile and young monkeys compared with adult animals. In contrast with the relatively low level of sequence divergence of B. quintana reported in humans, our investigation revealed much higher genetic diversity in nonhuman primates. We identified 44 new nucleotide variable sites and 14 novel sequence types (STs) among the B. quintana isolates by MLST analysis. Some STs were found only in cynomolgus macaques, while some others were detected only in rhesus macaques, suggesting evidence of host‐cospeciation, which were further confirmed by phylogenetic analysis and Splits decomposition analysis. Our findings suggest that trench fever may primarily be a zoonotic disease with macaques as the natural hosts.     

River islands,refugia and genetic structuring in the endemic brown frog Rana kukunoris (Anura,Ranidae) of the Qinghai‐Tibetan Plateau

Frequently, Pleistocene climatic cycling has been found to be the diver of genetic structuring in populations, even in areas that did not have continental ice sheets, such as on the Qinghai‐Tibetan Plateau (QTP). Typically, species distributed on the plateau have been hypothesized to re‐treat to south‐eastern refugia, especially during the Last Glacial Maximum (LGM). We evaluated sequence variation in the mitochondrial DNA gene Cytb and the nuclear DNA gene RAG‐1 in Rana kukunoris, a species endemic to the QTP. Two major lineages, N and S, were identified, and lineage N was further subdivided into N1 and N2. The geographical distribution and genealogical divergences supported the hypothesis of multiple refugia. However, major lineages and sublineages diverged prior to the LGM. Demographical expansion was detected only in lineage S and sublineage N2. Sublineage N1 might have survived several glacial cycles in situ and did not expand after the LGM because of the absence of suitable habitat; it survived in river islands. Genetic analysis and environment modelling suggested that the north‐eastern edge of QTP contained a major refugium for R. kukunoris. From here, lineage S dispersed southwards after the LGM. Two microrefugia in northern Qilian Mountains greatly contributed to current level of intraspecific genetic diversity. These results were found to have important implications for the habitat conservation in Northwest China.     

Mechanism of random integration of foreign DNA in transgenic mice

Little is known about how foreign DNA is randomly integrated into chromosomes in transgenic animals. In the current study, the insertion sites of 36 transgenic mice were mapped by thermal asymmetric interlaced PCR, and 38 junction sequences were obtained from 30 samples. Analysis of the 38 sequences revealed that 44.7 % of integration events occurred within host gene regions, including 13.2 % (5/38) in exonic regions and 31.6 % (12/38) in intronic regions. The results also revealed that all non-end side integrations of foreign DNA were mediated by short sequence homologies (microhomologies) and that the end side integrations occurred in the presence or absence of microhomologies. In addition, microhomology-mediated mechanisms were also confirmed in four transgenic Arabidopsis thaliana lines. The results indicate that foreign DNA is easily integrated into host gene regions. These results also suggest that the integration of both ends of foreign DNA follows the above-mentioned mechanism in many transgenic/transformed organisms.     

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.     

Synthesis and biological evaluation of 18F labeled fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives for sigma-1 receptor imaging

We report the synthesis and evaluation of a series of fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives as potential σ₁ receptor ligands. In vitro competition binding assays showed that 1-(1,3-benzodioxol-5-ylmethyl)-4-(4-(2-fluoroethoxy)benzyl)piperazine (6) exhibits low nanomolar affinity for σ₁ receptors (K_i = 1.85 ± 1.59 nM) and high subtype selectivity (σ₂ receptor: K_i = 291 ± 111 nM; K_iσ₂/K_iσ₁ = 157). [¹⁸F]6 was prepared in 30–50% isolated radiochemical yield, with radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic ¹⁸F^? substitution of the corresponding tosylate precursor. The log D_{pH 7.4} value of [¹⁸F]6 was found to be 2.57 ± 0.10, which is within the range expected to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of radiotracers in organs known to contain σ₁ receptors, including the brain, lungs, kidneys, heart, and spleen. Administration of haloperidol 5 min prior to injection of [¹⁸F]6 significantly reduced the concentration of radiotracers in the above-mentioned organs. The accumulation of radiotracers in the bone was quite low suggesting that [¹⁸F]6 is relatively stable to in vivo defluorination. The ex vivo autoradiography in rat brain showed high accumulation of radiotracers in the brain areas known to possess high expression of σ₁ receptors. These findings suggest that [¹⁸F]6 is a suitable radiotracer for imaging σ₁ receptors with PET in vivo.