Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase

Bacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis (Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5'-monophosphate (dTMP) efficiently with K (m) and V (max) values of 33 microM and 48 micromol mg(-1) min(-1), respectively. The efficiency of deoxyuridine-5'-monophosphate phosphorylation was approximately 10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2'-fluoroarabinosyl-5-methyldeoxyuridine-5'-monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but L-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5'-monophosphate (5FdUMP) and 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4TMP). No activity could be detected with 3'-azidothymidine-5'-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and d-FMAU was a strong inhibitor with an IC(50) value of 10 microM, while other nucleosides--L-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2'-arabinosylthymidine--were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents.     

Synthesis of resveratrol analogues, and evaluation of their cytotoxic and xanthine oxidase inhibitory activities

Thirteen resveratrol (=5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol) analogues with a CHO group have been prepared by partial synthesis from resveratrol. The synthesized compounds have been evaluated for their cytotoxic activity against a human nasopharyngeal epidermoid tumor cell line KB, as well as for their xanthine oxidase inhibitory activity. Compounds 2, 3, and 6a showed the most significant cytotoxic activities against the cell line KB, and compound 2 also exhibited strong xanthine oxidase inhibitory activity.     

Chimeric analogs of human β-defensin 1 and θ-defensin disrupt pre-established bacterial biofilms

Antibiofilm activity of several human defensin analogs that have the ability to kill planktonic bacteria, against pre-established biofilms of Escherichia coli MG1655 and Staphylococcus aureus NCTC 8530 were examined. Linear and linear fatty acylated analogs did not show any activity while disulfide constrained analogs disrupted pre-established S. aureus biofilms. Chimeric analogs of human β-defensin 1 and θ-defensin, hBTD-1 and [d]hBTD-1 were highly active against S. aureus biofilms. Among the analogs tested, only the d-enantiomer [d]hBTD-1 showed activity against E. coli biofilm. Our study provides insights into the structural requirements for the eradication of pre-established biofilms in defensin analogs.     

Modified biphenyl Hsp90 C-terminal inhibitors for the treatment of cancer

Heat Shock Protein 90 (Hsp90) is a molecular chaperone under clinical investigation for the treatment of neurodegenerative diseases and cancer. Neuroprotective Hsp90 C-terminal inhibitors (novologues) contain a biaryl ring system, and include KU-596, which was modified and investigated for potential anti-cancer activity. Incorporation of a benzamide group onto the biaryl novologues in lieu of the acetamide yielded compounds that manifest anti-cancer activity. Further exploration of the central phenyl ring led to compounds with enhanced anti-proliferative activity. The design, synthesis, and evaluation of these new analogs against breast and prostate cancer cell lines is reported herein, where it was found that 8b and 10 manifest potent anti-proliferative activity and a robust degradation of Hsp90 client-dependent proteins.     

Fluoroethoxy-1,4-diphenethylpiperidine and piperazine derivatives: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2

A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine derivatives were designed, synthesized and evaluated for their ability to inhibit [³H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [³H]serotonin (5-HT) uptake at the serotonin transporter (SERT), and [³H]dofetilide binding at the human-ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition of [³H]DA uptake at VMAT2, Ki changes in the nanomolar range (K_i?=?0.014–0.073?µM). Compound 15d exhibited the highest affinity (K_i?=?0.014?µM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (K_i?=?0.073?µM). Compound 15b was considered the lead compound from this analog series due to its high affinity and selectivity for VMAT2.     

NMR structures and molecular dynamics simulation of hylin‐a1 peptide analogs interacting with micelles

Antimicrobial peptides are recognized candidates with pharmaceutical potential against epidemic emerging multi‐drug resistant bacteria. In this study, we use nuclear magnetic resonance spectroscopy and molecular dynamics simulations to determine the unknown structure and evaluate the interaction with dodecylphosphatidylcholine (DPC) and sodium dodecylsulphate (SDS) micelles with three W⁶‐Hylin‐a1 analogs antimicrobial peptides (HyAc, HyK, and HyD). The HyAc, HyK, and HyD bound to DPC micelles are all formed by a unique α‐helix structure. Moreover, all peptides reach the DPC micelles' core, which thus suggests that the N‐terminal modifications do not influence the interaction with zwiterionic surfaces. On the other hand, only HyAc and HyK peptides are able to penetrate the SDS micelle core while HyD remains always at its surface. The stability of the α‐helical structure, after peptide‐membrane interaction, can also be important to the second step of peptide insertion into the membrane hydrophobic core during permeabilization. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Pyrimidine non-nucleoside analogs: A direct synthesis of a novel class of N-substituted amino and N-sulfonamide derivatives of pyrimidines

A convenient method for the regioselective synthesis of pyrimidine non-nucleoside analogs was developed. This study reports a novel and efficient method for the synthesis of a new type of N-substituted amino methylsulfanylpyrimidines and the corresponding pyrazolo[3,4-d]pyrimidines. This series of compounds was designed through the reaction of dimethyl N-cyanodithioiminocarbonate with 2-cyano-N′-(thiophen-2-yl-, furan-2-yl- and pyridin-4-ylmethylene)acetohydrazide and N′-(2-cyanoacetyl)arylsulfonohydrazides. The scope and limitation of the method are demonstrated. The antibacterial and antifungal activities of the synthesized compounds were also evaluated.     

Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation.

In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxyguanosine kinase (dCK/dGK)-like enzymes, which are deoxycytidine and/or purine deoxyribonucleoside-specific kinases. We cloned and characterized two new deoxyribonucleoside kinases belonging to the TK2-like group from the insect Bombyx mori and the amphibian Xenopus laevis. The deoxyribonucleoside kinase from B. mori (Bm-dNK) turned out to be a multisubstrate kinase like Dm-dNK. But uniquely for a deoxyribonucleoside kinase, Bm-dNK displayed positive cooperativity with all four natural deoxyribonucleoside substrates. The deoxyribonucleoside kinase from X. laevis (Xen-PyK) resembled closely the human and mouse TK2 enzymes displaying their characteristic Michaelis-Menten kinetic with deoxycytidine and negative cooperativity with its second natural substrate thymidine. Bm-dNK, Dm-dNK and Xen-PyK were shown to be homodimers. Significant differences in the feedback inhibition by deoxyribonucleoside triphosphates between these three enzymes were found. The insect multisubstrate deoxyribonucleoside kinases Bm-dNK and Dm-dNK were only inhibited by thymidine triphosphate, while Xen-PyK was inhibited by thymidine and deoxycytidine triphosphate in a complex pattern depending on the deoxyribonucleoside substrate. The broad substrate specificity and different feedback regulation of the multisubstrate insect deoxyribonucleoside kinases may indicate that these enzymes have a different functional role than the other members of the TK2-like group.     

1alpha,25-dihydroxyvitamin D3 stimulates phosphorylation of IkappaBalpha and synergizes with TPA to induce nuclear translocation of NFkappaB during monocytic differentiation of NB4 leukemia cells.

Treatment of NB4 acute promyelocytic leukemia cells with 1,25-dihydroxyvitamin D3 (1,25D3) or analogs 20-epi-22-oxa-24a,26a,27a-trihomo-1alpha,25-dihydroxyvitamin D3, 1,24-dihydroxy-22-ene-24-cyclopropylvitamin D3, 1alpha,25-dihydroxylumisterol3, or 1alpha,25(OH)2-d5-previtamin D3 in combination with TPA induces monocytic differentiation. The role of 1,25D3 in the induction of maturation has been shown to be a priming effect. Differentiation in response to these agents requires VDR-independent signaling of 1,25D3, PKC signaling, intracellular calcium, and calpain activity. In this study we identify the NFkappaB/IkappaB signaling pathway as a target of 1,25D3 and TPA action. One of the priming effects of 1,25D3 appears to be the rapid phosphorylation of serine residues on IkappaBalpha. On their own, 1,25D3, its analogs, and TPA do not alter IkappaBalpha expression; however, combinations of analogs with TPA result in a synergistic decrease in IkappaBalpha expression. Decreased expression of IkappaBalpha likely results from enhanced degradation, which allows the observed subsequent nuclear translocation of NFkappaB subunit p65. Since nuclear-localized NFkappaB was observed only in combination-treated cells, it is proposed that nuclear targets of NFkappaB are required for monocytic differentiation. Intracellular calcium and proteolytic activity are both necessary for the induction of IkappaB regulation and translocation of NFkappaB and are critical components of the nongenomic signaling cascades of the 1,25D3-induced differentiation pathway.     

根据R基因保守区分离小麦R基因类似序列

用根据核苷酸结合位点（nucleotide binding site,NBS)和丝氨酸／苏氨酸蛋白质激酶域设计的2对简并性引物,以小麦－簇毛麦6VS／6AL易位系的cDNA为模板进行PCR扩增,扩增产物克隆到pGEM-T载体中,经测序,共获得具有NBS结构域特征的片段克隆9个和具有丝氨酸／苏氨酸蛋白质激酶域特征的片段的克隆1个。将克隆之间核苷酸序列同源性高于90％的克隆归为一类,把9个NBS片段分为6类。这6类抗病基因类似序列（resistance gene analogs,PGA)均具有阅读框,并与已克隆的小麦抗条锈病基因γr、大麦抗白粉病基因Mla1和Mla6、拟南芥的抗病基因RPS2以及其他一些抗病基因在NBS保守区内具有高度的同源性。用小麦中国春缺体－四体初步将它们分别定位于小麦第一、第二和第五部分同源群上,进一步用5′－RACE技术获得RGA N5的5′端,发现期编码产物的N端还具有6个亮氨酸拉链（leucine zipper,LZ),与RPS2的N－端有较高同源性。