Unique antibiotic sensitivity of archaebacterial polypeptide elongation factors.

The antibiotic sensitivity of the archaebacterial factors catalyzing the binding of aminoacyl-tRNA to ribosomes (elongation factor Tu [EF-Tu] for eubacteria and elongation factor 1 [EF1] for eucaryotes) and the translocation of peptidyl-tRNA (elongation factor G [EF-G] for eubacteria and elongation factor 2 [EF2] for eucaryotes) was investigated by using two EF-Tu and EF1 [EF-Tu(EF1)]-targeted drugs, kirromycin and pulvomycin, and the EF-G and EF2 [EF-G(EF2)]-targeted drug fusidic acid. The interaction of the inhibitors with the target factors was monitored by using polyphenylalanine-synthesizing cell-free systems. A survey of methanogenic, halophilic, and sulfur-dependent archaebacteria showed that elongation factors of organisms belonging to the methanogenic-halophilic and sulfur-dependent branches of the "third kingdom" exhibit different antibiotic sensitivity spectra. Namely, the methanobacterial-halobacterial EF-Tu(EF1)-equivalent protein was found to be sensitive to pulvomycin but insensitive to kirromycin, whereas the methanobacterial-halobacterial EF-G(EF2)-equivalent protein was found to be sensitive to fusidic acid. By contrast, sulfur-dependent thermophiles were unaffected by all three antibiotics, with two exceptions; Thermococcus celer, whose EF-Tu(EF1)-equivalent factor was blocked by pulvomycin, and Thermoproteus tenax, whose EF-G(EF2)-equivalent factor was sensitive to fusidic acid. On the whole, the results revealed a remarkable intralineage heterogeneity of elongation factors not encountered within each of the two reference (eubacterial and eucaryotic) kingdoms.     

Electron-paramagnetic-resonance studies of manganese(II) complexes with elongation factor Tu from Bacillus stearothermophilus. Observation of a GTP hydrolysis intermediate state complex

Changes in the coordination of Mn2+ to nucleotide, water and protein at the active site of elongation factor Tu (EF-Tu) have been studied by electron paramagnetic resonance (EPR) spectroscopy. From the time dependence of the Mn2+ spectrum after addition of GTP to EF-Tu X Mn, it was apparent that three complexes with different EPR linewidths could be detected. Using additional information from the kinetics of 32Pi production and release from EF-Tu X Mn X [gamma-32P]GTP these were identified as EF-Tu X Mn X GTP (linewidth 4.2 mT), EF-Tu X Mn X GDP X Pi (1.20 mT) and EF-Tu X Mn X GDP (1.29 mT). The linewidth for EF-Tu X Mn was 1.51 mT. The rate constant for GTP cleavage on EF-Tu was 0.01 min-1 at 24 C, for Pi release from the EF-Tu X GDP X Pi complex 0.0033 min-1. The corresponding rate constants in the presence of Mg2+ were 0.003 min-1 and 0.0065 min-1. The rate constant for reversal of the cleavage step was found to be much smaller than that for the rate of Pi release (and consequently much smaller than in the forward direction), as shown by 31P-NMR experiments on the incorporation of 18O into Pi from GTP hydrolyzed in the presence of H2 18O. EPR experiments using specifically 17O-labelled GTPs demonstrated an interaction of Mn2+ with the beta-phosphate in both the EF-Tu X GDP X Pi and EF-Tu X GDP complexes. Inorganic phosphate in the EF-Tu X GDP X Pi complex was found not to interact with the metal ion. From EPR experiments in H2 17O, it was concluded that the most probable number of water molecules in the different complexes was 4 (EF-Tu X Mn), 5 (EF-Tu X Mn X GDP X Pi) and 3 (EF-Tu X Mn X GDP), with 2, 0 and 2 metal-protein interactions respectively.     

1,3-disubstituted 4-aminopiperidines as useful tools in the optimization of the 2-aminobenzo[a]quinolizine dipeptidyl peptidase IV inhibitors

In a search for novel DPP-IV inhibitors, 2-aminobenzo[a]quinolizines were identified as submicromolar HTS hits. Due to the difficult synthetic access to this compound class, 1,3-disubstituted 4-aminopiperidines were used as model compounds for optimization. The developed synthetic methodology and the SAR could be transferred to the 2-aminobenzo[a]quinolizine series, leading to highly active DPP-IV inhibitors.     

Biosynthesis of diacylglyceryl-N,N,N-trimethylhomoserine in Rhodobacter sphaeroides and evidence for lipid-linked N methylation.

Rhodobacter sphaeroides, which produces diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) under phosphate-limiting conditions, was incubated with L-[1-14C]- and L-[methyl-14C]methionine in pulse and pulse-chase experiments. The label was incorporated specifically into the polar part of DGTS and of three other compounds. One of them (compound 3) could be identified as diacylglyceryl-N,N-dimethylhomoserine by cochromatography with a reference obtained semisynthetically from DGTS. It was labelled when using L-[1-14C]- as well as L-[methyl-14C]methionine as a precursor and was converted to DGTS when incubated with the DGTS-forming eukaryotic alga Ochromonas danica (Chrysophyceae). Of the other two compounds labelled with L-[1-14C]methionine, compound 2 was also labelled with L-[methyl-14C]methionine whereas compound 1 was not, suggesting that these two intermediates are the corresponding N-methyl and nonmethylated lipids, respectively. The methyltransferase inhibitor 3'-deazaadenosine enhanced the amounts of compounds 1 to 3 but decreased the amount of DGTS. It is concluded that in R. sphaeroides, DGTS is synthesized by the same pathway as in eukaryotic organisms and that the N methylation is the terminal step in this process and occurs on the preformed lipid. Since the phosphatidylcholine-deficient mutant CHB20, lacking the phosphatidylcholine-forming N-methyltransferase was able to synthesize DGTS, one or several separate N-methyltransferases are suggested to be responsible for the synthesis of DGTS.     

Selective inhibitors of GABA uptake: synthesis and molecular pharmacology of 4-N-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol analogues

A series of lipophilic diaromatic derivatives of the glia-selective GABA uptake inhibitor (R)-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol [(R)-exo-THPO, 4] were synthesized via reductive amination of 3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazol-4-one (9) or via N-alkylation of O-alkylatedracemic 4. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation or primary cultures of mouse cortical neurons and glia cells (astrocytes), as well as HEK cells transfected with cloned mouse GABA transporter subtypes (GAT1-4). The activity against isoniazid-induced convulsions in mice after subcutaneous administration of the compounds was determined. All of the compounds were potent inhibitors of synaptosomal uptake the most potent compound being (RS)-4-[N-(1,1-diphenylbut-1-en-4-yl)amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (17a, IC50 = 0.14 microM). The majority of the compounds showed a weak preference for glial, as compared to neuronal, GABA uptake. The highest degree of selectivity was 10-fold corresponding to the glia selectivity of (R)-N-methyl-exo-THPO (5). All derivatives showed a preference for the GAT1 transporter, as compared with GAT2-4, with the exception of (RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (28d), which quite surprisingly turned out to be more potent than GABA at both GAT1 and GAT2 subtypes. The GAT1 activity was shown to reside in (R)-28d whereas (R)-28d and (S)-28d contributed equally to GAT2 activity. This makes (S)-28d a GAT2 selective compound, and (R)-28d equally effective in inhibition of GAT1 and GAT2 mediated GABA transport. All compounds tested were effective as anticonvulsant reflecting that these compounds have blood-brain barrier permeating ability.     

Crystal structure, solid state and solution characterisation of copper(II) coordination compounds of ethyl 5-methyl-4-imidazolecarboxylate (emizco)

The synthesis and characterisation of the following compounds derived from the biological relevant compound ethyl 5-methyl-4-imidazolecarboxylate (emizco) (1): [Cu(emizco)Cl2] (2), [Cu(emizco)2Cl2] (3), [Cu(emizco)2Br2] (4), [Cu(emizco)2(H2O)2](NO3)2 (5) and [Cu(emizco)4](NO3)2 (6), is presented. These compounds were characterised by IR and UV spectroscopic techniques, in addition the crystal structures of compounds 1-5 were determined. For complexes 2-5, emizco is coordinated as a bidentate ligand, through the oxygen atom of the carboxylate moiety and the nitrogen atom of the imidazolic ring. Different geometries are stabilised: compound 2 includes a pentacoordinated square pyramidal metal centre, while 3-5 are derived from octahedral geometry. Halide compounds 3 and 4 show a cis-octahedral arrangement, which is not very common on [CuN2O2X2] systems, while 5 stabilises the trans-octahedral isomer. Compound 6 displays a square planar geometry. Finally, hydrolysis of emizco to its corresponding carboxylic acid (mizco), allowed the preparation of another square planar complex 7, identified as [Cu(mizco)2] 0.5H2O. Solution studies of these compounds indicate that emizco is not substituted from the coordination sphere, remaining as a bidentate ligand. Halides are substituted by water molecules, changing from cis octahedral to the trans-[Cu(emizco)2(H2O)2]2+ isomer.     

Design, synthesis, and pharmacological evaluation of new neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives with in vivo hypnotic and analgesic profile

The present study describes the synthesis and pharmacological profiles of four novel pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives 2-5, which were structurally designed by using the sedative and analgesic drug zolpidem 1 as lead compound. The heterotricyclic system present in the target compounds 2-5 was constructed in good yields, exploiting a regioselective hetero Diels-Alder reaction of the key azabutadiene derivative 7 and functionalized N-phenylmaleimides 9-12. Additionally, we identified that 1-methyl-7-(4-nitrophenyl)-3-phenyl-3,6,7,8-tetrahydropyrazolo[3,4-b]pyrrolo[3,4-d]pyridine-6,8-dione derivative (LASSBio-873, 5) presented not only the most potent ability to promote sedation, which was similar to that induced by the standard benzodiazepine drug midazolam, but also potent central antinociceptive effect.     

紫茎泽兰一种潜在化感活性物质的分离鉴定

【背景】紫茎泽兰潜在的化感效应可能是其具有极强入侵性的重要原因。目前,针对紫茎泽兰潜在化感活性物质的研究主要集中于偏小极性的萜烯类化合物,而对大极性化学物质的研究相对较少。【方法】采用活性跟踪分析法对紫茎泽兰乙酸乙酯提取物大极性亚组分进行潜在化感活性物质的跟踪分离,并用现代波谱技术解析其化学结构,最后就分离获得的单体成分与已报道的化感活性物质羟基泽兰酮（2）和泽兰二酮（3）以及信号分子JA-Ile在抑制拟南芥种子萌发和幼苗根生长方面进行潜在化感活性的对比分析。【结果】分离鉴定出一个具有潜在强化感活性的大极性化合物2．香豆酸葡萄糖苷（1）,研究发现该化合物虽然对拟南芥种子萌发的抑制作用不显著,但在0．1mmol·L^-1的浓度条件下对拟南芥幼苗根生长却有着比羟基泽兰酮和泽兰二酮更为显著的抑制活性,且其抑制作用的浓度一活性关系与信号分子JA-Ile类似。【结论与意义】紫茎泽兰中潜在大极性的非萜烯类化感活性物质对于紫茎泽兰的综合化感效应可能同样具有重要而不可或缺的作用,化合物1等潜在大极性化感活性物质的逐步揭示对于系统解析紫茎泽兰的化感效应机制具有重要意义。     

Systematic analysis of the pharmacological function of Schisandra as a potential exercise supplement

[Purpose] Exercise can prevent conditions such as atrophy and degenerative brain diseases. However, owing to individual differences in athletic ability, exercise supplements can be used to improve a person’s exercise capacity. Schisandra chinensis (SC) is a natural product with various physiologically active effects. In this study, we analyzed SC using a pharmacological network and determined whether it could be used as an exercise supplement.[Methods] The active compounds of SC and target genes were identified using the Traditional Chinese Medicine Database and Analysis Platform (TCMSP). The active compound and target genes were selected based on pharmacokinetic (PK) conditions (oral bioavailability (OB) ≥ 30%, Caco-2 permeability (Caco-2) ≥ -0.4, and drug-likeness (DL) ≥ 0.18). Gene ontology (GO) was analyzed using the Cytoscape software.[Results] Eight active compounds were identified according to the PK conditions. Twenty-one target genes were identified after excluding duplicates in the eight active compounds. The top 10 GOs were analyzed using GO-biological process analysis. GO was subsequently divided into three representative categories: postsynaptic neurotransmitter receptor activity (53.85%), an intracellular steroid hormone receptor signaling pathway (36.46%), and endopeptidase activity (10%). SC is related to immune function.[Conclusion] According to the GO analysis, SC plays a role in immunity and inflammation, promotes liver metabolism, improves fatigue, and regulates the function of steroid receptors. Therefore, we suggest SC as an exercise supplement with nutritional and anti-fatigue benefits.     

Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803

Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H₂O₂, whereas GDP-bound EF-Tu was resistant to H₂O₂. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H₂O₂, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.     

Isolation and characterization of Streptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state

The ability of EF-Tu to aggregate spontaneously was employed for the purification of homogeneous EF-Tu . GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF-Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19,100 +/- 1,000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U). Aggregated EF-Tu . GDP exhibited almost eight-times lower GDP-exchange capacity at 2 degrees C than at 30 degrees C. This suggests that GDP-binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF-Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF-Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP-binding sites.     

Elongation factor Ts can act as a steric chaperone by increasing the solubility of nucleotide binding-impaired elongation factor-Tu.

Several elongation factor (EF) Tu mutants (T25A, H22Y/T25S, D80N, D138N) that have impaired nucleotide binding show decreased solubility on overexpression in the E. coli cell, an indication that they do not fold correctly. Moreover, EF-Tu[T25A] and EF-Tu[D80N] were shown to inhibit cell growth on expression, an effect attributed to their sequestration of EF-Ts [Krab, I. M., and Parmeggiani, A. (1999) J. Biol. Chem. 274, 11132--11138; Krab, I. M., and Parmeggiani, A. (1999) Biochemistry 38, 13035--13041]. We present here results showing that the co-overexpression of EF-Ts at a 1:1 ratio dramatically improves the solubility of mutant EF-Tu, although in the case of EF-Tu[D138N]--which cannot at all bind the nucleotides available in the cell--this is a slow process. Moreover, with co-overexpression of EF-Ts, the mentioned growth inhibition is relieved. We conclude that for the formation of a correct EF-Tu structure the nucleotide plays an important role as a "folding nucleus", and also that in its absence EF-Ts can act as a folding template or steric chaperone for the correct folding of EF-Tu.     

In vitro characterization of a gamma-secretase radiotracer in mammalian brain

Inhibition of gamma-secretase is a potential therapeutic target for Alzheimer's disease (AD). The present studies have characterized the in vitro properties of a radiolabeled small molecule gamma-secretase inhibitor, [3H]compound D (Yan et al., 2004, J. Neurosci.24, 2942-2952) in mammalian brain. [3H]Compound D was shown to bind with nanomolar affinity (Kd = 0.32-1.5 nM) to a single population of saturable sites in rat, rhesus and human brain cortex homogenates, the density of binding sites ranging from 4 to 7 nM across the species. Competition studies with a structurally diverse group of gamma-secretase inhibitors with a wide range of binding affinities showed that the binding affinities of these compounds correlated well with their ability to inhibit gamma-secretase in vitro. Autoradiographic studies showed that the specific binding of [3H]compound D was widely distributed throughout adult rat, rhesus and normal human brain. There did not appear to be any difference in distribution of [3H]compound D specific binding sites in AD cortex compared with control human cortex as measured using tissue section autoradiography, nor any correlation between gamma-secretase binding and plaque burden as measured immunohistochemically. [3H]compound D is a useful tool to probe the expression and pharmacology of gamma-secretase in mammalian brain.     

Enzymatic hydrolysis of green tea seed extract and its activity on 5alpha-reductase inhibition

Two kaempferol glycosides were isolated from green tea seed extract (GTSE). After conducting a structure analysis, these two compounds were identified as kaempferol-3-O-[2-O-beta-D-galactopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside (compound 1) and kaempferol-3-O-[2-O-beta-D-xylopyranosyl-6-O-alpha-L-rhanmopyranosyl]-beta-D-glucopyranoside (compound 2). These two compounds were hydrolysed by o-glycolytic enzymes for the production of kaempferol. After performing several reactions, we found the optimum enzyme combination, a reaction with beta-galactosidase and hesperidinase. Finally, we produced kaempferol of above 95% purity. The 5alpha-reductase inhibition activities of GTSE hydrolysate (GTSE-H) containing kaempferol were evaluated by the contact cell-based metabolic method using a stable HEK 293 cell line. GTSE-H showed a good inhibition effect on HEK 293 cell lines both type 1 and type 2 on 5alpha-reductase. Especially, GTSE-H inhibited type 2 with kaempferol content dependency. The results indicate that the inhibition activity of hydrolysate on 5alpha-reductase type 2 increases in accordance with kaempferol content.     

The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of elongation factor Tu. The functional and structural role of residue Cys81.

A Cys residue located in the second consensus sequence element (DCPG) of the GTP-binding region is highly conserved in bacterial elongation factors (EF) Tu. Chemical modification of this Cys81 in EF-Tu from Escherichia coli by N-tosyl-L-phenylalanine chloromethane [Jonák, J., Petersen, T. E., Clark, B. F. C. & Rychlík, I. (1982) FEBS Lett. 150, 485-488], and of homologous Cys residues in other bacterial EF-Tu, selectively blocks the binding of Xaa-tRNA. We have substituted Cys81 with Gly using site-directed mutagenesis of the EF-Tu-encoding tuf A gene. This substitution induces a partial inhibition (20-70%) of: (a) poly(U)-directed poly(Phe) synthesis; (b) EF-Tu/Xaa-tRNA interaction, determined as protection by EF-Tu of the non-enzymic deacylation of Xaa-tRNA; (c) EF-Tu-dependent binding of Xaa-tRNA to the mRNA/ribosome complex and (d) the intrinsic GTPase reaction, that is also less sensitive to stimulation by Xaa-tRNA. Our results thus provide evidence that Cys81, though important, is not essential for the binding of Xaa-tRNA to EF-Tu. The accuracy in poly(Phe) synthesis, measured as misincorporation of Leu, was increased. Both the binding affinity of [C81G]EF-Tu for the nucleotide and the resistance against thermal denaturation are more strongly decreased in the case of the GDP-bound state than in the case of the GTP-bound state, suggesting that Cys81 plays a more specific role in the former conformation. The sensitivity to N-tosyl-L-phenylalanine chloromethane is decreased by 80% but not totally lost. The inhibition by N-tosyl-L-phenylalanine chloromethane treatment of the function of EF-Tu appears to be a consequence of steric hindrance and/or of an altered conformation of EF-Tu.GTP. The lower activities of [C81G]EF-Tu are probably due to long-range effects, mediated by an overall destabilization of the molecule that is particularly pronounced for the GDP-bound state.     

Identification of novel species-selective agonists of the G-protein-coupled receptor GPR35 that promote recruitment of β-arrestin-2 and activate Gα13

The poorly characterized G-protein-coupled receptor GPR35 has been suggested as a potential exploratory target for the treatment of both metabolic disorders and hypertension. It has also been indicated to play an important role in immune modulation. A major impediment to validation of these concepts and further study of the role of this receptor has been a paucity of pharmacological tools that interact with GPR35. Using a receptor-β-arrestin-2 interaction assay with both human and rat orthologues of GPR35, we identified a number of compounds possessing agonist activity. These included the previously described ligand zaprinast. Although a number of active compounds, including cromolyn disodium and dicumarol, displayed similar potency at both orthologues of GPR35, a number of ligands, including pamoate and niflumic acid, had detectable activity only at human GPR35 whereas others, including zaprinast and luteolin, were markedly selective for the rat orthologue. Previous studies have demonstrated activation of Gα13 by GPR35. A Saccharomyces cerevisiae-based assay employing a chimaeric Gpa1-Gα13 G-protein confirmed that all of the compounds active at human GPR35 in the β-arrestin-2 interaction assay were also able to promote cell growth via Gα13. Each of these ligands also promoted binding of [35S]GTP[S] (guanosine 5'-[γ-[35S]thio]triphosphate) to an epitope-tagged form of Gα13 in a GPR35-dependent manner. The ligands identified in these studies will be useful in interrogating the biological actions of GPR35, but appreciation of the species selectivity of ligands at this receptor will be vital to correctly attribute function.     

Screening of drug databank against WT and mutant main protease of SARS-CoV-2: Towards finding potential compound for repurposing against COVID-19

Although several pharmacological agents are under investigation to be repurposed as therapeutic against COVID-19, not much success has been achieved yet. So, the search for an effective and active option for the treatment of COVID-19 is still a big challenge. The Spike protein (S), RNA-dependent RNA polymerase (RdRp), and Main protease (Mpro) are considered to be the primary therapeutic drug target for COVID-19. In this study we have screened the drugbank compound library against the Main Protease. But our search was not limited to just Mpro. Like other viruses, SARS-CoV-2, have also acquired unique mutations. These mutations within the active site of these target proteins may be an important factor hindering effective drug candidate development. In the present study we identified important active site mutations within the SARS-CoV-2 Mpro (Y54C, N142S, T190I and A191V). Further the drugbank database was computationally screened against Mpro and the selected mutants. Finally, we came up with the common molecules effective against the wild type (WT) and all the selected Mpro. The study found Imiglitazar, was found to be the most active compound against the wild type of Mpro. While PF-03715455 (Y54C), Salvianolic acid A (N142S and T190I), and Montelukast (A191V) were found to be most active against the other selected mutants. It was also found that some other compounds such as Acteoside, 4-Amino-N- {4-[2-(2,6-Dimethyl-Phenoxy)-Acetylamino]-3-Hydroxy-1-Isobutyl-5-Phenyl-Pentyl}-Benzamide, PF-00610355, 4-Amino-N-4-[2-(2,6-Dimethyl-Phenoxy)-Acetylamino]-3-Hydroxy-1-Isobutyl-5-Phenyl-Pentyl}-Benzamide and Atorvastatin were showing high efficacy against the WT as well as other selected mutants. We believe that these molecules will provide a better and effective option for the treatment of COVID-19 clinical manifestations.     

Synthesis and anti-tumor activity of [1,4] dioxino [2,3-f] quinazoline derivatives as dual inhibitors of c-Met and VEGFR-2

Both c-Met and VEGFR-2 were important targets for cancer therapies. In order to develop reversible and non-covalent c-Met and VEGFR-2 dual inhibitors, a series of [1,4]dioxino[2,3-f]quinazoline derivatives were designed and synthesized. The enzyme assay demonstrated that most target compounds had inhibition potency on both c-Met and VEGFR-2 with IC₅₀ values in nanomolar range especially compounds 7m and 7k. Based on further cell proliferation assay in vitro, compound 7k showed significantly anti-tumor activity in vivo on a hepatocellular carcinoma (MHCC97H cells) xenograft mouse model. We docked the compound 7m with c-Met and VEGFR-2 kinases, and interpreted the SAR of these analogues. All results indicated that the target compounds were dual inhibitors of c-Met and VEGFR-2 kinases that held promising potential in cancer therapy.     

Aminoacyl-tRNA exclusively in the 3'-isomeric form is bound to polypeptide chain elongation factor Tu

2' and 3'-O-(N-acetyl-L-phenylalanyl)adenosine (Ac-Phe-Ado) were chemically synthesized. These two isomers were clearly separated from each other by high-performance liquid chromatography (HPLC). From the two isomers of [3H]Phe-tRNA in equilibrium, Ac-[3H]Phe-Ado was prepared, without any change in the 2'/3'-isomer ratio, by acetylation of the phenylalanyl residue with acetic anhydride followed by digestion with pancreatic RNase A. By HPLC analysis of this preparation of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was found to be 0.20:0.80. Further, [3H]Phe-tRNA was bound to Escherichia coli polypeptide chain elongation factor Tu (EF-Tu) with the ligand of GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GMP-P(NH)P). The ternary complex was treated with phenol and acetic anhydride, and then digested with pancreatic RNase A. By HPLC analysis of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was determined to be 0.07:0.93 in the complex with EF-Tu.GTP and 0.04:0.96 in the complex with EF-Tu.GMP-P(NH)P. These results clearly indicate that the 3'-isomer, rather than the 2'-isomer, of aminoacyl-tRNA is exclusively involved in the ternary complex.