Fragment-based lead discovery of indazole-based compounds as AXL kinase inhibitors.

AXL is a member of the TAM (TYRO3, AXL, MER) subfamily of receptor tyrosine kinases. It is upregulated in a variety of cancers and its overexpression is associated with poor disease prognosis and acquired drug resistance. Utilizing a fragment-based lead discovery approach, a new indazole-based AXL inhibitor was obtained. The indazole fragment hit 11, identified through a high concentration biochemical screen, was expeditiously improved to fragment 24 by screening our in-house expanded library of fragments (ELF) collection. Subsequent fragment optimization guided by docking studies provided potent inhibitor 54 with moderate exposure levels in mice. X-ray crystal structure of analog 50 complexed with the I650M mutated kinase domain of Mer revealed the key binding interactions for the scaffold. The good potency coupled with reasonable kinase selectivity, moderate in vivo exposure levels, and availability of structural information for the series makes it a suitable starting point for further optimization efforts.

Toxicity and effects of four insecticides on Na+, K+-ATPase of western flower thrips, Frankliniella occidentalis.

Western flower thrips (WFT), Frankliniella occidentalis, has become an important pest of vegetables worldwide, due to its economic damage to crop production. In order to control WFT, chemical insecticides are widely used. However, WFT has developed a high resistance against many kinds of insecticides. Na+, K+-ATPase, playing an important role in the ionic transmission across the membrane, is commonly considered to be the target of several xenobiotic compounds. However, whether the Na+, K+-ATPase can be used as one of the target sites for controlling WFT is still unknown. In this study, resistance levels of WFT to four insecticides (chlorpyrifos, beta cypermethrin, abamectin, and thiamethoxam) were measured. It was found that all four insecticides exhibited significant inhibitory effects on WFT, especially on nymphs. The activity of Na+, K+-ATPase was estimated after the treatment of four insecticides. Additionally, mRNA expression levels of three Na+, K+-ATPase α-subunit isoforms (X1, X2 and X3) were detected using RT-qPCR. The transcription profile of three Na+, K+-ATPase α-subunit isoforms were diverse after treatment by these four insecticides, which indicated that these isoforms might play different roles in the tolerance to insecticides. The results suggested that Na+, K+-ATPase can obviously be inhibited by these four classes of insecticide, and may serve as the new target for controlling WFT.

Characterization of ductal carcinoma in situ cell lines established from breast tumor of a Singapore Chinese patient.

BACKGROUND: Five cell lines were established from a Singaporean patient of Chinese origin with breast ductal carcinoma in situ (DCIS). These five cell lines express exogenous human telomerase reverse transcriptase (hTERT) which confers the ability to proliferate indefinitely. METHODS: Cells were isolated from the DCIS excision and transfected with a plasmid expressing hTERT, a catalytic subunit of telomerase. Five immortalized colonies were propagated and characterized by karyotyping, array comparative genomic hybridization (CGH), immunostaining and Western blots for biomarkers, in vitro anchorage independent growth, in vivo mouse tumorigenicity, drug sensitivity, species authentication and virology safety testing. RESULTS: Array CGH analysis showed that the cell lines harbored different specific genetic aberrations. Common mutations observed in most breast cancer cell lines such as the general loss of heterozygosity (LOH) throughout chromosome X and chromosome 17 are also observed in our cell lines. The cell lines were further characterized as human breast cells that are estrogen- and progesterone-receptor positive, and sensitive to tamoxifen. The cell lines showed anchorage-independent growth in the soft agar assay and can grow in common culture medium without supplementation with growth factor, therefore demonstrating transformed characteristics. Four of the cell lines can engraft and form measureable tumors after 50 days when injected subcutaneously into immune-deficient (SCID) mice. The weak tumorigenicity of these cell lines corresponded well with their non-malignant growth origin. The cell lines were authenticated to be of human origin based on DNA fingerprint of the cells. The cell lines were free from contamination of 20 viruses and mycoplasma in the virological safety test panel. CONCLUSIONS: Unlike most available breast cell lines, our cell lines are derived from primary breast cancer tissues that represent earlier grades or tumor progression stages. They would be useful as study models for basic and clinical research programs directed at early diagnosis and intervention.

Cytotoxic activity and cell specificity of a novel LHRH peptide drug conjugate, D-Cys6-LHRH vedotin, against ovarian cancer cell lines.

Ovarian cancer is the most deadly female gynaecological malignancy in developed countries and new treatments are urgently needed. The luteinising hormone releasing hormone (LHRH) peptide drug conjugate Zoptarelin doxorubicin is one such potential new drug modality that entered clinical trials for treating LHRH receptor-positive gynaecological cancers. However, development stopped after disappointing Phase 3 results in 2017. We believe the lack of efficacy was due to linker instability and payload potency. In this work, we replaced its linker-toxin with vedotin (MC-VC-PABC-MMAE), yielding the novel peptide drug conjugate D-Cys6-LHRH vedotin. A GI50 and cell specificity comparison against cancerous and non-cancerous ovarian cell lines showed significantly superior bioactivity and selectivity over Zoptarelin doxorubicin (GI50 4 vs. 453 nM) and other chemotherapeutic drugs used for treating ovarian cancers. Our results suggest D-Cys6-LHRH vedotin can potentially be used as a treatment for ovarian cancer.

The elevation of salinity above 1% deteriorated nitrification performance and reshaped nitrifier community of an MBR: An often overlooked factor in the treatment of high-strength ammonium wastewater.

The effects of synchronous variations of influent salinity with the elevation of NH4+-N concentration on nitrification performance and microbial community structure of bioreactor are often ignored. In this study, we investigated the dynamic response of nitrifying activated sludge to synchronously increased salinity and ammonia loading rate (ALR) in a nitrification membrane bioreactor (MBR). We found that the increase in influent salinity above 1% (from 0.91 to 1.32%) led to the deterioration of the nitrification performance of the MBR. The combined inhibition effect of salinity (1.32%), free ammonia (FA, an average of 1.37 mg/L), and free nitrous acid (FNA, an average of 0.155 mg/L) on nitrite-oxidizing bacteria (NOB) resulted in long-term (35 days) nitrite accumulation. The further increase of salinity and ALR exhibited little influence on the nitrification performance of MBR after the activated sludge had adapted to high salinity (>1%), effective nitrification performance was achieved at high ALR up to 1.71 kg NH4+-N/m3·d and high salinity (2.13%). The microbial analysis showed that the elevated salinity and accumulation of FNA reshaped the microbial community structure of ammonia-oxidizing bacteria (AOB) and NOB. The dominant species of AOB and NOB shifted from the salinity-resistant species Nitrosomonas aestuarii to the species Nitrosomonas mobilis with dual resistant to salinity and FNA, and from non-salinity-resistant species Candidatus Nitrospira defluvii to salinity-resistant species Nitrobacter winogradskyi and Nitrospira marina, respectively. Therefore, the salinity of 1% may be a critical level for the nitrification performance and the shift in the nitrifier community of activated sludge without salinity acclimation.

Structure and size determination of bacteriophage P2 and P4 procapsids: function of size responsiveness mutations.

Bacteriophage P4 is dependent on structural proteins supplied by a helper phage, P2, to assemble infectious virions. Bacteriophage P2 normally forms an icosahedral capsid with T=7 symmetry from the gpN capsid protein, the gpO scaffolding protein and the gpQ portal protein. In the presence of P4, however, the same structural proteins are assembled into a smaller capsid with T=4 symmetry. This size determination is effected by the P4-encoded protein Sid, which forms an external scaffold around the small P4 procapsids. Size responsiveness (sir) mutants in gpN fail to assemble small capsids even in the presence of Sid. We have produced large and small procapsids by co-expression of gpN with gpO and Sid, respectively, and applied cryo-electron microscopy and three-dimensional reconstruction methods to visualize these procapsids. gpN has an HK97-like fold and interacts with Sid in an exposed loop where the sir mutations are clustered. The T=7 lattice of P2 has dextro handedness, unlike the laevo lattices of other phages with this fold observed so far.

The complete mitochondrial genome and phylogenetic analysis of Tipula (Yamatotipula) nova Walker, 1848 (Diptera, Tipulidae) from Qingdao, Shandong, China.

The genus Tipula Linnaeus, 1758 is a large group of crane flies with more than 2400 known species from 41 subgenera. In this study, we report the first complete mitochondrial (mt) genome sequence of the subgenus Tipula (Yamatotipula), which is a circular molecule of 15,668 bp with an AT content of 77.2%. The mt genome contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a long non-coding region. Three conserved overlapping regions, 8 bp between tRNATrp and tRNACys , 7 bp between ATP8 and ATP6, and 7 bp between ND4 and ND4L, are found. Phylogenetic analysis reveals that the Tipulomorpha includes the family Trichoceridae and the Trichoceridae is sister-group to the remaining Tipulomorpha.

Bidirectional interactions between beet armyworm and its host in response to different fertilization conditions.

Fertilizer with different ratios of nitrogen (N) to phosphorus (P) can influence crop plant performance and defense against herbivores. Spodoptera exigua is an important agricultural pest that has caused serious economic loss, especially in recent decades. In the present study, we explored effects of different intensities and durations of S. exigua herbivory on host plant biomass and on S. exigua enzyme activities in response to five fertilizer treatments with different N: P ratios of 1: 5, 1: 3, 1: 1, 3: 1 and 5: 1. The results showed that fertilizer type can significantly influence interactions between caterpillars and its hosts. Compensatory growth of leaf biomass was detected under fertilizer with N: P = 3: 1. Fertilizer with a higher proportion of N appears to maintain stem biomass in defoliated seedlings similar to controls that are not exposed to herbivory. There was no significant difference in root biomass under most conditions. High proportion of N also enhanced the activity of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD) in low density of beet armyworm. However, with increased herbivorous intensity, a higher proportion of P played a more important role in increasing the activities of CAT and SOD. Higher P likely enhanced acetylcholine esterase (AChE) activity at lower degrees of defoliation, but a higher N proportion resulted in higher AChE activity at higher degrees of defoliation. Higher N proportion contributed to reduced carboxylesterase (CarE) activity at high intensity, short-term defoliation. However, when defoliation intensity increased, the difference in CarE activity between fertilizer categories was little. The study explored the interaction between the damage of S. exigua and the biomass accumulation of its host plant Brassica rapa, and the influence of the N/P ratio in plant fertilizer on this interaction. Systematic analysis was provided on the biomass of B. rapa and the activity of metabolic enzymes of S. exigua under different treatments.

The nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its N-terminal domain and multimerization properties.

The coronavirus nucleocapsid (N) protein packages viral genomic RNA into a ribonucleoprotein complex. Interactions between N proteins and RNA are thus crucial for the assembly of infectious virus particles. The 45 kDa recombinant nucleocapsid N protein of coronavirus infectious bronchitis virus (IBV) is highly sensitive to proteolysis. We obtained a stable fragment of 14.7 kDa spanning its N-terminal residues 29-160 (IBV-N29-160). Like the N-terminal RNA binding domain (SARS-N45-181) of the severe acute respiratory syndrome virus (SARS-CoV) N protein, the crystal structure of the IBV-N29-160 fragment at 1.85 A resolution reveals a protein core composed of a five-stranded antiparallel beta sheet with a positively charged beta hairpin extension and a hydrophobic platform that are probably involved in RNA binding. Crosslinking studies demonstrate the formation of dimers, tetramers, and higher multimers of IBV-N. A model for coronavirus shell formation is proposed in which dimerization of the C-terminal domain of IBV-N leads to oligomerization of the IBV-nucleocapsid protein and viral RNA condensation.

West Nile virus core protein; tetramer structure and ribbon formation.

We have determined the crystal structure of the core (C) protein from the Kunjin subtype of West Nile virus (WNV), closely related to the NY99 strain of WNV, currently a major health threat in the U.S. WNV is a member of the Flaviviridae family of enveloped RNA viruses that contains many important human pathogens. The C protein is associated with the RNA genome and forms the internal core which is surrounded by the envelope in the virion. The C protein structure contains four alpha helices and forms dimers that are organized into tetramers. The tetramers form extended filamentous ribbons resembling the stacked alpha helices seen in HEAT protein structures.

The cumulative damage index method: a new method for evaluating the effectiveness of control measures for Plutella xylostella (Lepidoptera: Plutellidae).

BACKGROUND: All previously and currently used methods for effectiveness evaluation of control measures for the diamondback moth (DBM) do not simultaneously take the actual damage and population size into consideration. Here, we propose a new method, the cumulative damage index method, in which the number of larvae and their amount of food consumption are simultaneously included in the calculation of the theoretical cumulative damage index (T) and actual cumulative damage index (A). Evaluation was based on the reduced degree of damage calculated according to indexes T and A. RESULTS: Based on the new method, the corrected effectiveness of the combined use of biological measures, chemical insecticides, Bacillus thuringiensis (Bt) and P. xylostella granulosis virus (PxGV) on DBM was 35.85, 2.37, 12.50 and 11.77% respectively. Under the action of natural factors, the Population Developmental Index (I) of DBM was 5.1 ± 1.4; under the integrated actions of natural factors and these four types of measure, index I of DBM was 0.34 ± 0.1, 6.1 ± 1.5, 2.1 ± 0.5 and 1.1 ± 0.3 respectively. The sole effectiveness of Trichogramma spp. when integrated with other natural factors, integrated biological measures and chemical insecticides was 21.43 ± 1.69%, 45.27 ± 4.09% and 20.68 ± 2.60% respectively. CONCLUSIONS: There was some difference between the effectiveness evaluated by the new method and index I, and the actual damage caused by DBM could be reflected well by index A. The new method is more scientifically appropriate and practical for effectiveness evaluation than existing methods. © 2014 Society of Chemical Industry.

Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-XL.

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Small molecule inhibitors of Bcl-X(L) function have been discovered from diverse structure classes using rational drug design as well as high-throughput screening (HTS) approaches. However, most of the BH3 mimetics that have been identified via screening based on fluorescence polarization displayed an affinity for their presumed protein targets that is far lower than that of BH3-only proteins. Therefore, it is important to establish a simple and inexpensive secondary platform for hit validation which is pertinent to current efforts for developing compounds that mimic the action of BH3-only proteins as novel anticancer agents. These considerations prompted us to explore the differential scanning fluorimetry (DSF) method that is based on energetic coupling between ligand binding and protein unfolding. We have systematically tested known Bcl-X(L)/Bcl-2 inhibitors using DSF and have revealed distinct subsets of inhibitors. More importantly, we report that some of these inhibitors interacted selectively with glutathione S-transferase tagged Bcl-X(L), whereas certain inhibitors exhibited marked selectivity towards native untagged Bcl-X(L). Therefore, we propose that the affinity tag may cause a significant conformational switch in the Bcl-X(L), which results in the selectivity for certain subsets of small molecule inhibitors. This finding also implies that the previous screens involving tagged proteins need to be carefully reexamined while further investigations must ensure that the right conformation of protein is used in future screens.

Structure of the equine arteritis virus nucleocapsid protein reveals a dimer-dimer arrangement.

Equine arteritis virus (EAV) is an enveloped positive-sense RNA virus belonging to the Arteriviridae family, which also includes the porcine pathogen PRRSV and is genetically and structurally related to the coronaviruses. EAV is an important equine pathogen that has caused significant economic losses to the horse-breeding industry and has been difficult to control. The EAV virion consists of a genome-containing nucleocapsid core made of nucleocapsid (N) protein surrounded by a lipid envelope containing several membrane proteins. Here, the crystal structure of the capsid-forming domain of the EAV N protein is presented at 2.0 A resolution. The dimeric N-protein structure is similar to the previously determined structure of the N protein from PRRSV, with most differences localized to the terminal helices and flexible loops. The N protein is organized as dimers of dimers in the crystal, which may reflect the arrangement of the protein in the viral nucleocapsid.

Assembly of bacteriophage P2 and P4 procapsids with internal scaffolding protein.

Assembly of the E. coli bacteriophage P2 into an icosahedral capsid with T = 7 symmetry is dependent on the gpN capsid protein, the gpQ connector protein and the gpO internal scaffolding protein. In the presence of the P4-encoded protein Sid, the same proteins are assembled into a smaller capsid with T = 4 symmetry. Although gpO has long been expected to act as an internal scaffolding protein, it has not been possible to produce P2 procapsids efficiently in vitro or in vivo due to a failure to express gpO at high levels. In this study, we find that full-length gpO undergoes proteolytic degradation within 1 h of induction of expression. However, a truncated version of gpO lacking the N-terminal 25 amino acids (Odelta25) is stably expressed at high levels and is able to direct the formation of P2 size procapsids. In the presence of Sid, Odelta25 is incorporated into P4 procapsids, showing that Sid overrides the effect of gpO on capsid size determination.

Amino acids 1055 to 1192 in the S2 region of severe acute respiratory syndrome coronavirus S protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents.

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SDelta10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

The structure of P4 procapsids produced by coexpression of capsid and external scaffolding proteins.

The double-stranded DNA bacteriophage P4 has a T = 4 icosahedral arrangement of the gpN capsid protein derived from the P2 helper phage. The precursor procapsids in addition contain an external scaffold made up of the P4-encoded Sid protein. High yields of pure P4 procapsids have been obtained by coexpressing the gpN and Sid proteins from a chimeric plasmid. Biochemical measurements show that the ratio of gpN to Sid in the procapsids is 2:1, corresponding to 120 copies of Sid per procapsid particle. A reconstruction of the P4 procapsid, made from 213 particle images to an effective resolution of about 21 A, greatly improves on the previously determined P4 procapsid structures. The structure shows a T = 4 capsid shell and a unique tandem arrangement of 120 copies of chilli-shaped Sid monomers, which form trimers and dimers on the procapsid surface.

Cleavage leads to expansion of bacteriophage P4 procapsids in vitro.

Proteolytic cleavage of the structural proteins is an important part of the maturation process for most bacteriophages and other viruses. In the double-stranded DNA bacteriophages this cleavage is associated with DNA packaging, capsid expansion, and scaffold removal. To understand the role of protein cleavage in the expansion of bacteriophages P2 and P4, we have experimentally cleaved P4 procapsids produced by overexpression of the capsid and scaffolding proteins. The cleavage leads to particle expansion and scaffold removal in vitro. The resulting expanded capsid has a thin-shelled structure similar, but not identical, to that of mature virions.