Synthesis and In Vitro Evaluation of C-7 and C-8 Luteolin Derivatives as Influenza Endonuclease Inhibitors.

The part of the influenza polymerase PA subunit featuring endonuclease activity is a target for anti-influenza therapies, including the FDA-approved drug Xofluza. A general feature of endonuclease inhibitors is their ability to chelate Mg or Mn ions located in the enzyme's catalytic site. Previously, we screened a panel of flavonoids for PA inhibition and found luteolin and its C-glucoside orientin to be potent inhibitors.

Structural and Thermodynamic Analysis of the Resistance Development to Pimodivir (VX-787), the Clinical Inhibitor of Cap Binding to PB2 Subunit of Influenza A Polymerase.

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap.

Structure and function of naturally evolved de novo proteins.

Comparative evolutionary genomics has revealed that novel protein coding genes can emerge randomly from non-coding DNA. While most of the myriad of transcripts which continuously emerge vanish rapidly, some attain regulatory regions, become translated and survive. More surprisingly, sequence properties of de novo proteins are almost indistinguishable from randomly obtained sequences, yet de novo proteins may gain functions and integrate into eukaryotic cellular networks quite easily.

Putative ligand binding sites of two functionally characterized bark beetle odorant receptors.

Background: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors.

structural characterization of the interaction between the C-terminal domain of the influenza polymerase PA subunit and an optimized small peptide inhibitor.

Influenza viruses can cause severe respiratory infections in humans, leading to nearly half a million deaths worldwide each year. Improved antiviral drugs are needed to address the threat of development of novel pandemic strains. Current therapeutic interventions target three key proteins in the viral life cycle: neuraminidase, the M2 channel and RNA-dependent-RNA polymerase.

Unraveling the anti-influenza effect of flavonoids: Experimental validation of luteolin and its congeners as potent influenza endonuclease inhibitors.

The biological effects of flavonoids on mammal cells are diverse, ranging from scavenging free radicals and anti-cancer activity to anti-influenza activity. Despite appreciable effort to understand the anti-influenza activity of flavonoids, there is no clear consensus about their precise mode-of-action at a cellular level. Here, we report the development and validation of a screening assay based on AlphaScreen technology and illustrate its application for determination of the inhibitory potency of a large set of polyols against PA N-terminal domain (PA-Nter) of influenza RNA-dependent RNA polymerase featuring endonuclease activity.

DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors.

Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains.

Copper-mediated arylsulfanylations and arylselanylations of pyrimidine or 7-deazapurine nucleosides and nucleotides.

The syntheses of 5-arylsulfanyl- or 5-arylselanylpyrimidine and 7-arylsulfanyl- or 7-arylselanyl-7-deazapurine nucleosides and nucleotides were developed by the Cu-mediated sulfanylations or selanylations of the corresponding 5-iodopyrimidine or 7-iodo-7-deazapurine nucleosides or nucleotides with diaryldisulfides or -diselenides. The reactions were also applicable for direct modifications of 2'-deoxycytidine triphosphate and the resulting 5-arylsulfanyl or 5-arylselanyl-dCTP served as substrates for the polymerase synthesis of modified DNA bearing arylsulfanyl or arylselanyl groups in the major groove.

Structural studies of the yeast DNA damage-inducible protein Ddi1 reveal domain architecture of this eukaryotic protein family.

The eukaryotic Ddi1 family is defined by a conserved retroviral aspartyl protease-like (RVP) domain found in association with a ubiquitin-like (UBL) domain. Ddi1 from Saccharomyces cerevisiae additionally contains a ubiquitin-associated (UBA) domain. The substrate specificity and role of the protease domain in the biological functions of the Ddi family remain unclear.

Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation.

Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomography provided structures of immature and mature HIV-1 with a diameter of 120-140 nm, but information about the sequence and dynamics of structural rearrangements is lacking.

Human DNA-Damage-Inducible 2 Protein Is Structurally and Functionally Distinct from Its Yeast Ortholog.

Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor.

Synthesis and evaluation of 2-pyridinylpyrimidines as inhibitors of HIV-1 structural protein assembly.

In an effort to identify an HIV-1 capsid assembly inhibitor with improved solubility and potency, we synthesized two series of pyrimidine analogues based on our earlier lead compound N-(4-(ethoxycarbonyl)phenyl)-2-(pyridine-4-yl)quinazoline-4-amine. In vitro binding experiments showed that our series of 2-pyridine-4-ylpyrimidines had IC50 values higher than 28μM. Our series of 2-pyridine-3-ylpyrimidines exhibited IC50 values ranging from 3 to 60μM.

Kinetic, thermodynamic and structural analysis of tamiphosphor binding to neuraminidase of H1N1 (2009) pandemic influenza.

Influenza virus causes severe respiratory infections that are responsible for up to half a million deaths worldwide each year. Two inhibitors targeting viral neuraminidase have been approved to date (oseltamivir, zanamivir). However, the rapid development of antiviral drug resistance and the efficient transmission of resistant viruses among humans represent serious threats to public health.

Synthesis, structural characterization, docking, lipophilicity and cytotoxicity of 1-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-3-alkyl carbamates, novel acetylcholinesterase and butyrylcholinesterase pseudo-irreversible inhibitors.

In the current study, sixteen novel derivatives of (R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethanamine were synthesized as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. Chemical structures together with purity of the synthesized compounds were substantiated by IR, (1)H, (13)C, (19)F NMR, high resolution mass spectrometry and elemental analysis. The optical activities were confirmed by optical rotation measurements.

The SQM/COSMO filter: reliable native pose identification based on the quantum-mechanical description of protein-ligand interactions and implicit COSMO solvation.

Current virtual screening tools are fast, but reliable scoring is elusive. Here, we present the 'SQM/COSMO filter', a novel scoring function featuring a quantitative semiempirical quantum mechanical (SQM) description of all types of noncovalent interactions coupled with implicit COSMO solvation. We show unequivocally that it outperforms eight widely used scoring functions.

Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal Domain of the Capsid Protein: Evaluation of 2-Arylquinazolines as Potential Antiviral Compounds.

Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly.

Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7-Substituted 7-Deazaguanine Nucleobases.

Previous studies of polymerase synthesis of base-modified DNAs and their cleavage by restriction enzymes have mostly related only to 5-substituted pyrimidine and 7-substituted 7-deazaadenine nucleotides. Here we report the synthesis of a series of 7-substituted 7-deazaguanine 2'-deoxyribonucleoside 5'-O-triphosphates (dG(R) TPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type II restriction endonucleases (REs). The dG(R) TPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching.

The properties of substituted 3D-aromatic neutral carboranes: the potential for σ-hole bonding.

The calculated properties of substituted carboranes such as dipole moment, polarisability, the magnitude of the σ-hole and the desolvation free energy are compared with these properties in comparable aromatic and cyclic aliphatic organic compounds. Dispersion and charge transfer energies are similar. However, the predicted strength of the halogen bonds with the same electron donor (based on the magnitude of the σ-hole) is larger for neutral C-vertex halogen-substituted carboranes than for their organic counterparts.

Enzymological description of multitemplate PCR-Shrinking amplification bias by optimizing the polymerase-template ratio.

Multitemplate polymerase chain reaction (PCR) is used for preparative and analytical applications in diagnostics and research. Classical PCR and qPCR are two basic setups with many possible experimental modifications. Classical PCR is a method of choice to obtain enough material for subsequent sophisticated applications such as construction of libraries for next-generation sequencing or high-throughput screening.

Catalytic Cycle of Multicopper Oxidases Studied by Combined Quantum- and Molecular-Mechanical Free-Energy Perturbation Methods.

We have used combined quantum mechanical and molecular mechanical free-energy perturbation methods in combination with explicit solvent simulations to study the reaction mechanism of the multicopper oxidases, in particular, the regeneration of the reduced state from the native intermediate. For 52 putative states of the trinuclear copper cluster, differing in the oxidation states of the copper ions and the protonation states of water- and O2-derived ligands, we have studied redox potentials, acidity constants, isomerization reactions, as well as water- and O2 binding reactions. Thereby, we can propose a full reaction mechanism of the multicopper oxidases with atomic detail.

Preorganization of the catalytic Zn(2+)-binding site in the HNH nuclease motif--A solution study.

The structure of the active site in a metalloenzyme can be a key determinant of its metal ion binding affinity and catalytic activity. In this study, the conformational features of the Zn(2+)-binding HNH motif were investigated by CD-spectroscopy in combination with isothermal microcalorimetric titrations. Various point mutations, including T454A, K458A and W464A, were introduced into the N-terminal loop of the nuclease domain of colicin E7 (NColE7).

Retroviral proteases and their roles in virion maturation.

Proteolytic processing of viral polyproteins is essential for retrovirus infectivity. Retroviral proteases (PR) become activated during or after assembly of the immature, non-infectious virion. They cleave viral polyproteins at specific sites, inducing major structural rearrangements termed maturation.

Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor.

HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis.

Chalcogen and pnicogen bonds in complexes of neutral icosahedral and bicapped square-antiprismatic heteroboranes.

A systematic quantum mechanical study of σ-hole (chalcogen, pnicogen, and halogen) bonding in neutral experimentally known closo-heteroboranes is performed. Chalcogens and pnicogens are incorporated in the borane cage, whereas halogens are considered as exo-substituents of dicarbaboranes. The chalcogen and pnicogen atoms in the heteroborane cages have partial positive charge and thus more positive σ-holes.