Xenobiotic nucleic acids (XNA) are nucleic acid analogues not present in nature that can be used for the storage of genetic information. In vivo XNA applications could be developed into novel ...biocontainment strategies, but are currently limited by the challenge of developing XNA processing enzymes such as polymerases, ligases and nucleases. Here, we present a structure-guided modelling-based strategy for the rational design of those enzymes essential for the development of XNA molecular biology. Docking of protein domains to unbound double-stranded nucleic acids is used to generate a first approximation of the extensive interaction of nucleic acid processing enzymes with their substrate. Molecular dynamics is used to optimise that prediction allowing, for the first time, the accurate prediction of how proteins that form toroidal complexes with nucleic acids interact with their substrate. Using the Chlorella virus DNA ligase as a proof of principle, we recapitulate the ligase's substrate specificity and successfully predict how to convert it into an XNA-templated XNA ligase.
Feline infectious peritonitis (FIP) caused by feline coronavirus (FCoV) is a common dis-ease in cats, fatal if untreated, and no effective treatment is currently legally available. The aim of this ...study was to evaluate efficacy and toxicity of the multi-component drug Xraphconn
in vitro and as oral treatment in cats with spontaneous FIP by examining survival rate, development of clinical and laboratory parameters, viral loads, anti-FCoV antibodies, and adverse effects. Mass spectrometry and nuclear magnetic resonance identified GS-441524 as an active component of Xraphconn
. Eighteen cats with FIP were prospectively followed up while being treated orally for 84 days. Values of key parameters on each examination day were compared to values before treatment initiation using linear mixed-effect models. Xraphconn
displayed high virucidal activity in cell culture. All cats recovered with dramatic improvement of clinical and laboratory parameters and massive reduction in viral loads within the first few days of treatment without serious adverse effects. Oral treatment with Xraphconn
containing GS-441524 was highly effective for FIP without causing serious adverse effects. This drug is an excellent option for the oral treatment of FIP and should be trialed as potential effective treatment option for other severe coronavirus-associated diseases across species.
Epilepsy is a chronic brain disorder characterized by recurrent seizures due to abnormal, excessive and synchronous neuronal activities in the brain. It affects approximately 65 million people ...worldwide, one third of which are still estimated to suffer from refractory seizures. Glutamic acid decarboxylase (GAD) that converts glutamate into GABA is a key enzyme in the dynamic regulation of neural network excitability. Importantly, clinical evidence shows that lowered GAD activity is associated with several forms of epilepsy which are often treatment resistant. In the present study, we synthetized and explored the possibility of using ethyl ketopentenoate (EKP), a lipid-permeable GAD-inhibitor, to induce refractory seizures in zebrafish larvae. Our results demonstrate that EKP evoked robust convulsive locomotor activities, excessive epileptiform discharges and upregulated c-fos expression in zebrafish. Moreover, transgenic animals in which neuronal cells express apoaequorin, a Ca
-sensitive bioluminescent photoprotein, displayed large luminescence signals indicating strong EKP-induced neuronal activation. Molecular docking data indicated that this proconvulsant activity resulted from the direct inhibition of both gad67 and gad65. Limited protective efficacy of tested anti-seizure drugs (ASDs) demonstrated a high level of treatment resistance of EKP-induced seizures. We conclude that the EKP zebrafish model can serve as a high-throughput platform for novel ASDs discovery.
Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative ...bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
There is a common interest for studying xeno-nucleic acid systems in the fields of synthetic biology and the origin of life, in particular, those with an engineered backbone and possessing novel ...properties. Along this line, we have investigated xylonucleic acid (XyloNA) containing a potentially prebiotic xylose sugar (a 3'-epimer of ribose) in its backbone. Herein, we report for the first time the synthesis of four XyloNA nucleotide building blocks and the assembly of XyloNA oligonucleotides containing all the natural nucleobases. A detailed investigation of pairing and structural properties of XyloNAs in comparison to DNA/RNA has been performed by thermal UV-melting, CD, and solution state NMR spectroscopic studies. XyloNA has been shown to be an orthogonal self-pairing system which adopts a slightly right-handed extended helical geometry. Our study on one hand, provides understanding for superior structure-function (-pairing) properties of DNA/RNA over XyloNA for selection as an informational polymer in the prebiotic context, while on the other hand, finds potential of XyloNA as an orthogonal genetic system for application in synthetic biology.
In vivo imaging of biological processes is an important asset of modern cell biology. Selectively reacting fluorophores herein are an important tool and click chemistry reactions take a large share ...in these events. 5-Ethynyl-2'-deoxyuridine (EdU) is well known for visualizing DNA replication, but does not show any selectivity for incorporation into DNA. Striving for specific visualization of virus replication, in particular HIV replication, a series of propargylated purine deoxynucleosides were prepared aiming for selective incorporation by HIV reverse transcriptase (RT). We here report on the synthesis and preliminary biological effects (cellular toxicity, HIV inhibitory effects, and feasibility of the click reaction) of these nucleoside analogues.
The synthesis, base‐pairing properties and in vitro and in vivo characteristics of 5‐methyl‐isocytosine (isoCMe) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic ...acid)–DNA(d) mosaic backbone, are described. The required h‐isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d‐isoG base mispairing follows the order T>G>C while the h‐isoG base mispairing follows the order G>C>T. The h‐ and d‐isoCMe bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoCMe misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base‐pair interpretation for the deoxyribose and hexitol isoCMe/isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality.
Synthesis and base‐pairing properties of 5‐methyl‐isocytosine and isoguanine nucleosides incorporated in a hexitol nucleic acid (HNA)–DNA mosaic backbone are described. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base (see scheme).
Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to ...the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain
-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the
-position, we obtained the
-glycosylated benzimidazole derivatives as the major products, resembling the respective purine
-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the
-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine
and the
-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of ...vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure-activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity.
The flavivirus family contains several important human pathogens, such as Zika virus (ZIKV), dengue, West Nile, and Yellow Fever viruses, that collectively lead to a large, global disease burden. ...Currently, there are no approved medicines that can target these viruses. The sudden outbreak of ZIKV infections in 2015⁻2016 posed a serious threat to global public health. While the epidemic has receded, persistent reservoirs of ZIKV infection can cause reemergence. Here, we have used X-ray crystallography-based screening to discover two novel sites on ZIKV NS3 helicase that can bind drug-like fragments. Both sites are structurally conserved in other flaviviruses, and mechanistically significant. The binding poses of four fragments, two for each of the binding sites, were characterized at atomic precision. Site A is a surface pocket on the NS3 helicase that is vital to its interaction with NS5 polymerase and formation of the flaviviral replication complex. Site B corresponds to a flexible, yet highly conserved, allosteric site at the intersection of the three NS3 helicase domains. Saturation transfer difference nuclear magnetic resonance (NMR) experiments were additionally used to evaluate the binding strength of the fragments, revealing dissociation constants (K
) in the lower mM range. We conclude that the NS3 helicase of flaviviruses is a viable drug target. The data obtained open opportunities towards structure-based design of first-in-class anti-ZIKV compounds, as well as pan-flaviviral therapeutics.
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