Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

• Klíčová slova
• capsid, nucleolus, nucleus, protein structure, tick-borne flaviviruses, translational shutoff,
• MeSH
• kapsida metabolismus   MeSH
• RNA virová metabolismus   MeSH
• virové nestrukturální proteiny metabolismus   MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• viry klíšťové encefalitidy * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA virová MeSH
• virové nestrukturální proteiny MeSH
• virové plášťové proteiny MeSH

Bacterial methionine biosynthesis can take place by either the trans-sulfurylation route or direct sulfurylation. The enzymes responsible for trans-sulfurylation have been characterized extensively because they occur in model organisms such as Escherichia coli. However, direct sulfurylation is actually the predominant route for methionine biosynthesis across the phylogenetic tree. In this pathway, most bacteria use an O-acetylhomoserine aminocarboxypropyltransferase (MetY) to catalyze the formation of homocysteine from O-acetylhomoserine and bisulfide. Despite the widespread distribution of MetY, this pyridoxal 5'-phosphate-dependent enzyme remains comparatively understudied. To address this knowledge gap, we have characterized the MetY from Thermotoga maritima (TmMetY). At its optimal temperature of 70 °C, TmMetY has a turnover number (apparent kcat = 900 s-1) that is 10- to 700-fold higher than the three other MetY enzymes for which data are available. We also present crystal structures of TmMetY in the internal aldimine form and, fortuitously, with a β,γ-unsaturated ketimine reaction intermediate. This intermediate is identical to that found in the catalytic cycle of cystathionine γ-synthase (MetB), which is a homologous enzyme from the trans-sulfurylation pathway. By comparing the TmMetY and MetB structures, we have identified Arg270 as a critical determinant of specificity. It helps to wall off the active site of TmMetY, disfavoring the binding of the first MetB substrate, O-succinylhomoserine. It also ensures a strict specificity for bisulfide as the second substrate of MetY by occluding the larger MetB substrate, cysteine. Overall, this work illuminates the subtle structural mechanisms by which homologous pyridoxal 5'-phosphate-dependent enzymes can effect different catalytic, and therefore metabolic, outcomes.

• Klíčová slova
• X-ray crystallography, enzyme kinetics, enzyme mechanism, methionine, protein structure,
• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• biosyntetické dráhy MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• methionin metabolismus   MeSH
• molekulární modely MeSH
• Thermotoga maritima chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• methionin MeSH

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites' viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.

• MeSH
• hypoxanthinfosforibosyltransferasa antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• katalytická doména MeSH
• lidé MeSH
• metabolické sítě a dráhy účinky léků   MeSH
• molekulární modely MeSH
• objevování léků MeSH
• pentosyltransferasy antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• purinony metabolismus   MeSH
• RNA interference MeSH
• trypanocidální látky chemie   farmakologie   MeSH
• Trypanosoma brucei brucei účinky léků   enzymologie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hypoxanthine-guanine-xanthine phosphoribosyltransferase MeSH Prohlížeč
• hypoxanthinfosforibosyltransferasa MeSH
• inhibitory enzymů MeSH
• pentosyltransferasy MeSH
• purinony MeSH
• trypanocidální látky MeSH

Neuronal nitric oxide synthase (nNOS) is a target for development of antineurodegenerative agents. Most nNOS inhibitors mimic l-arginine and have poor bioavailability. 2-Aminoquinolines showed promise as bioavailable nNOS inhibitors but suffered from low human nNOS inhibition, low selectivity versus human eNOS, and significant binding to other CNS targets. We aimed to improve human nNOS potency and selectivity and reduce off-target binding by (a) truncating the original scaffold or (b) introducing a hydrophilic group to interrupt the lipophilic, promiscuous pharmacophore and promote interaction with human nNOS-specific His342. We synthesized both truncated and polar 2-aminoquinoline derivatives and assayed them against recombinant NOS enzymes. Although aniline and pyridine derivatives interact with His342, benzonitriles conferred the best rat and human nNOS inhibition. Both introduction of a hydrophobic substituent next to the cyano group and aminoquinoline methylation considerably improved isoform selectivity. Most importantly, these modifications preserved Caco-2 permeability and reduced off-target CNS binding.

• MeSH
• aminochinoliny chemická syntéza   farmakologie   MeSH
• Caco-2 buňky MeSH
• enzymatické testy MeSH
• histidin chemie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• skot MeSH
• synthasa oxidu dusnatého, typ I antagonisté a inhibitory   chemie   MeSH
• synthasa oxidu dusnatého, typ II antagonisté a inhibitory   MeSH
• synthasa oxidu dusnatého, typ III antagonisté a inhibitory   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• 4-(2-(((2-amino-4-methylquinolin-7-yl)methyl)amino)ethyl)-2-methylbenzonitrile MeSH Prohlížeč
• aminochinoliny MeSH
• histidin MeSH
• NOS1 protein, human MeSH Prohlížeč
• Nos1 protein, rat MeSH Prohlížeč
• Nos2 protein, mouse MeSH Prohlížeč
• synthasa oxidu dusnatého, typ I MeSH
• synthasa oxidu dusnatého, typ II MeSH
• synthasa oxidu dusnatého, typ III MeSH

Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that when two of the ANPs bind, they induce an unusual conformation change to the loop where the reaction product, pyrophosphate, is expected to bind. This and other structural differences between the Tbr and human enzymes suggest selective inhibitors for the Tbr enzyme can be designed.

• MeSH
• druhová specificita MeSH
• hypoxanthinfosforibosyltransferasa antagonisté a inhibitory   chemie   genetika   MeSH
• inhibitory enzymů farmakologie   MeSH
• katalytická doména MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• protozoální proteiny antagonisté a inhibitory   chemie   genetika   MeSH
• rekombinantní proteiny chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• trypanocidální látky farmakologie   MeSH
• Trypanosoma brucei brucei účinky léků   enzymologie   genetika   MeSH
• Trypanosoma cruzi enzymologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hypoxanthinfosforibosyltransferasa MeSH
• inhibitory enzymů MeSH
• protozoální proteiny MeSH
• rekombinantní proteiny MeSH
• trypanocidální látky MeSH

Neuronal nitric oxide synthase (nNOS) is an important therapeutic target for the treatment of various neurodegenerative disorders. A major challenge in the design of nNOS inhibitors focuses on potency in humans and selectivity over other NOS isoforms. Here we report potent and selective human nNOS inhibitors based on the 2-aminopyridine scaffold with a central pyridine linker. Compound 14j, the most promising inhibitor in this study, exhibits excellent potency for rat nNOS (Ki = 16 nM) with 828-fold n/e and 118-fold n/i selectivity with a Ki value of 13 nM against human nNOS with 1761-fold human n/e selectivity. Compound 14j also displayed good metabolic stability in human liver microsomes, low plasma protein binding, and minimal binding to cytochromes P450 (CYPs), although it had little to no Caco-2 permeability.

• MeSH
• aminopyridiny chemická syntéza   chemie   farmakologie   MeSH
• inhibitory enzymů chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• molekulární struktura MeSH
• synthasa oxidu dusnatého, typ I antagonisté a inhibitory   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• alpha-aminopyridine MeSH Prohlížeč
• aminopyridiny MeSH
• inhibitory enzymů MeSH
• synthasa oxidu dusnatého, typ I MeSH

Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to (a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions and (b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged scaffold significantly reduces off-target binding.

• MeSH
• aminochinoliny chemie   farmakokinetika   farmakologie   MeSH
• Caco-2 buňky MeSH
• fenylethery chemie   farmakokinetika   farmakologie   MeSH
• inhibitory enzymů chemie   farmakokinetika   farmakologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• synthasa oxidu dusnatého, typ I antagonisté a inhibitory   metabolismus   MeSH
• synthasa oxidu dusnatého, typ II antagonisté a inhibitory   metabolismus   MeSH
• synthasa oxidu dusnatého, typ III antagonisté a inhibitory   metabolismus   MeSH
• synthasa oxidu dusnatého antagonisté a inhibitory   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminochinoliny MeSH
• fenylethery MeSH
• inhibitory enzymů MeSH
• phenyl ether MeSH Prohlížeč
• synthasa oxidu dusnatého, typ I MeSH
• synthasa oxidu dusnatého, typ II MeSH
• synthasa oxidu dusnatého, typ III MeSH
• synthasa oxidu dusnatého MeSH

RNA polymerase in bacteria is a multisubunit protein complex that is essential for gene expression. We have identified a new subunit of RNA polymerase present in the high-A+T Firmicutes phylum of Gram-positive bacteria and have named it ε. Previously ε had been identified as a small protein (ω1) that copurified with RNA polymerase. We have solved the structure of ε by X-ray crystallography and show that it is not an ω subunit. Rather, ε bears remarkable similarity to the Gp2 family of phage proteins involved in the inhibition of host cell transcription following infection. Deletion of ε shows no phenotype and has no effect on the transcriptional profile of the cell. Determination of the location of ε within the assembly of RNA polymerase core by single-particle analysis suggests that it binds toward the downstream side of the DNA binding cleft. Due to the structural similarity of ε with Gp2 and the fact they bind similar regions of RNA polymerase, we hypothesize that ε may serve a role in protection from phage infection.

• MeSH
• Bacillus subtilis enzymologie   MeSH
• DNA řízené RNA-polymerasy chemie   genetika   metabolismus   MeSH
• fylogeneze MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• podjednotky proteinů MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u bakterií MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• podjednotky proteinů MeSH