Synthesis and anti-HCMV activity of 1-[ω-(phenoxy)alkyl]uracil derivatives and analogues thereof

HCMV infection represents a life-threatening condition for immunocompromised patients and newborn infants and novel anti-HCMV agents are clearly needed. In this regard, a series of 1-[ω-(phenoxy)alkyl]uracil derivatives were synthesized and examined for antiviral properties. Compounds 17, 20, 24 and 28 were found to exhibit highly specific and promising inhibitory activity against HCMV replication in HEL cell cultures with EC₅₀ values within 5.5–12 μM range. Further studies should be undertaken to elucidate the mechanism of action of these compounds and the structure–activity relationship for the linker region.     

Biochemical characterization and structure–function relationship of two plant NCS2 proteins,the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent K_M values were determined with 10.12 μM, 4.85 μM and 19.95 μM, respectively for NAT3 and 1.74 μM, 2.44 μM and 29.83 μM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton–nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1–7 and TM8–14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure–function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.     

In vivo engineering of proteins with nitrogen-containing tryptophan analogs

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.The term noncanonical amino acid refers to an amino acid that does not belong, in contrast to a canonical amino acid, to the genetically encoded, proteinogenic amino acids. The term analog defines a strict isosteric exchange of a canonical/noncanonical amino acid (e.g., tryptophan/azatryptophan), while the term surrogate defines a nonisosteric change (e.g., tryptophan/azulene). Mutant denotes a protein in which the wild-type sequence was changed by site-directed mutagenesis (codon manipulation on the DNA level) within the repertoire of the standard amino acids. Variant denotes a protein in which one or more canonical amino acids derived from a wild-type or a mutant sequence were replaced by a noncanonical one (expanded amino acid repertoire, codon reassignment on the protein translation level).     

Sugar-Driven Prebiotic Synthesis of 3,5(6)-Dimethylpyrazin-2-one: A Possible Nucleobase of a Primitive Replication Process

Reaction of glyceraldehyde with alanine amide (or ammonia) under anaerobic aqueous conditions yielded 3,5(6)-dimethylpyrazin-2-one that is considered a possible complementary residue of a primitive replicating molecule that preceded RNA. Synthesis of the dimethylpyrazin-2-one isomers under mild aqueous conditions (65 degrees C, pH 5.5) from 100 mM glyceraldehyde and alanine amide (or ammonia) was complete in about 5 days. This synthesis using 25 mM glyceraldehyde and alanine amide gave a total pyrazinone yield of 9.3% consisting of 42% of the 3,5-dimethylprazin-2-one isomer and 58% of the 3,6-dimethylpyrazin-2-one isomer. The related synthesis of the dimethylpyrazin-2-one isomers from glyceraldehyde and ammonia was about 200-fold less efficient than the alanine amide reaction. This synthetic process is considered a reasonable model of origin-of-life chemistry because it uses plausible prebiotic substrates, and resembles modern biosynthesis by employing the energized carbon groups of sugars to drive the synthesis of small organic molecules. Possible sugar-driven pathways for the prebiotic synthesis of polymerizable 2-pyrazinone monomers are discussed.     

Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote Aspergillus nidulans

Abstract

Despite detailed genetic and mutagenic analysis and a recent high-resolution structure of a bacterial member of the nucleobase-ascorbate transporter (NAT) family, understanding of the mechanism of action of eukaryotic NATs is limited. Preliminary studies successfully expressed and purified wild-type UapA to high homogeneity; however, the protein was extremely unstable, degrading almost completely after 48 h at 4°C. In an attempt to increase UapA stability we generated a number of single point mutants (E356D, E356Q, N409A, N409D, Q408E and G411V) previously shown to have reduced or no transport activity, but correct targeting to the membrane. The mutant UapA constructs expressed well as GFP fusions in Saccharomyces cerevisiae and exhibited similar fluorescent size exclusion chromatography (FSEC) profiles to the wild-type protein, following solubilization in 1% DDM, LDAO or OM + 1 mM xanthine. In order to assess the relative stabilities of the mutants, solubilized fractions prepared in 1% DDM + 1 mM xanthine were heated at 45°C for 10 min prior to FSEC. The Q408E and G411V mutants gave markedly better profiles than either wild-type or the other mutants. Further FSEC analysis following solubilization of the mutants in 1% NG ± xanthine confirmed that G411V is more stable than the other mutants, but showed that Q408E is unstable under these conditions. G411V and an N-terminally truncated construct G411VΔ1-11 were submitted to large-scale expression and purification. Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies.     

The ENT family of eukaryote nucleoside and nucleobase transporters: recent advances in the investigation of structure/function relationships and the identification of novel isoforms

The first examples of the equilibrative nucleoside transporter (ENT) family were characterized in human tissues at the molecular level only 4 years ago. Since that time, the identification of homologous proteins by functional cloning and genome analysis has revealed that the family is widely distributed in eukaryotes. Family members are predicted to possess 11 transmembrane helices (TMs), and recent investigations on the mammalian ENTs have implicated the TM 3-6 region in solute recognition. Whilst the name of the family reflects the properties ofits prototypical member hENT1, an equilibrative transporter of nucleosides, some family members can also transport nucleobases and some are proton-dependent, concentrative transporters. In addition to their role in nucleoside salvage, ENTs are targets for coronary vasodilator drugs and act as routes for uptake of cytotoxic drugs in humans and protozoa. This paper summarizes current knowledge of the family and reports on the identification of a novel mammalian ENT isoform, designated ENT3, from mouse and human tissues.     

Potential bioisosteres of β-uracilalanines derived from 1H-1,2,3-triazole-C-carboxylic acids

The 1H-1,2,3-triazole-originated derivatives of willardiine were obtained by: (i) construction of the 1H-1,2,3-triazole ring in 1,3-dipolar cycloaddition of the uracil-derived azides and the carboxylate-bearing alkynes or α-acylphosphorus ylide, or (ii) N-alkylation of the uracil derivative with the 1H-1,2,3-triazole-4-carboxylate-derived mesylate. The latter method offered: (i) reproducible results, (ii) a significant reduction of amounts of auxiliary materials, (iii) reduction in wastes and (iv) reduction in a number of manual operations required for obtaining the reaction product. Compound 6a exhibited significant binding affinity to hHS1S2I ligand-binding domain of GluR2 receptor (EC₅₀ = 2.90 µM) and decreased viability of human astrocytoma MOG-G-CCM cells in higher extent than known AMPA antagonist GYKI 52466.     

On the Physical Basis of the Amino Acid Polar Requirement

Understanding how codons became associated with their specific amino acids is fundamental to deriving a theory for the origin of the genetic code. Carl Woese and coworkers designed a series of experiments to test associations between amino acids and nucleobases that may have played a role in establishing the genetic code. Through these experiments it was found that a property of amino acids called the polar requirement (PR) is correlated with the organization of the codon table. No other property of amino acids has been found that correlates with the codon table as well as PR, indicating that PR is uniquely related to the modern genetic code. Using molecular dynamics simulations of amino acids in solutions of water and dimethylpyridine used to experimentally measure PR, we show that variations in the partitioning between the two phases as described by radial distribution functions correlate well with the measured PRs. Partition coefficients based on probability densities of the amino acids in each phase have the linear behavior with base concentration as suggested by PR experiments.     

Cyclic tetranuclear half-sandwich ruthenium(II) complexes with 4,7-phenanthroline and hydroxo bridges: crystal structure, solution behaviour and binding to nucleosides

The reaction between [(η⁶-p-cymene)Ru(H₂O)₃]X₂ and 4,7-phenanthroline (phen) leads to the formation of the rectangular tetranuclear complexes [(η⁶-p-cymene)₄Ru₄(μ-4,7-phen-N⁴,N⁷)₂(μ-OH)₄]X₄ (X = NO₃, 1a; SO₃CF₃, 1b) which have been structurally characterised by X-ray crystallography. ¹H NMR spectroscopic studies suggest the presence of a partially dissociated dinuclear species of type [(η⁶-p-cymene)₂Ru₂(μ-4,7-phen-N⁴,N⁷)(solv)₄]⁴⁺ in equilibrium with the tetranuclear cyclic species found in the solid state. The temperature effect for this equilibrium was studied by variable temperature ¹H NMR experiments in D₂O and MeOD. The results reveal that the proportion of the tetranuclear species increases with the polarity of the solvent which favour stacking interactions between the phenanthroline moieties. In addition, the reactivity of the tetranuclear species towards the nucleosides guanosine (Guo), cytidine (Cyt), 2′-deoxythymidine (Thy) and 2′-deoxyadenosine (dAdo) has been monitored by ¹H NMR as a potential model for the interaction of the 1 species with the probable DNA target. The results reveal that the 1 systems are able to bind the nucleobases endocyclic nitrogen atoms of Guo Cyt, and dAdo.     

Trypanosoma brucei: expression of multiple purine transporters prevents the development of allopurinol resistance

Allopurinol is a hypoxanthine analogue used to treat Leishmania infections that also displays activity against the related parasite Trypanosoma brucei. We have investigated the ease by which resistance to this drug is established in Trypanosoma brucei brucei and correlated this to the mechanisms by which it is accumulated by the parasite. Long-term exposure of procyclic T. b. brucei to 3mM allopurinol did not induce resistance. This appears to be related to the fact that allopurinol was taken up through two distinct nucleobase transporters, H1 and H4, both with high affinity for the drug. The apparent Km for [3H]allopurinol transport by H4 (2.1+/-0.4 microM) was determined by expressing the encoding gene in Saccharomyces cerevisiae. Long-term allopurinol exposure did not change Km (hypoxanthine), Ki (allopurinol), or Vmax values of either H1 or H4 transporters and the cells retained their ability to proliferate with hypoxanthine as sole purine source. This study shows that transport-related resistance to purine antimetabolites is not easily induced in Trypanosoma spp. as long as uptake is mediated by multiple transporters.