Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

Two novel classes of fused azaisocytosine-containing congeners as promising drug candidates: Design, synthesis as well as in vitro, ex vivo and in silico studies.

Searching for new less toxic anticancer drug candidates is a big challenge from a medical point of view. The present investigation was aimed at describing two independent synthetic approaches based on isosteric replacements, spectroscopic characteristics, in vitro anticancer and ex vivo antihaemolytic activities of novel molecules (9-22) and correlations between their standardised lipophilicity indices, computed log Paverage values and pharmacokinetic descriptors. Two novel protocols for annelation of the triazinone template on hydrazinylideneimidazolidines (1-8) (showing a high reactivity towards electrophilic reagents, such as ethyl trifluoropyruvate and ethyl 3-methyl-2-oxobutyrate) were developed for the first time, giving rise to two original classes of highly conjugated azaisocytosine-containing molecules (9-16 and 17-22). Both syntheses proceeded under basic conditions to yield the most probable intermediates (e.g. hemiaminals and imines), which in refluxing two-component solvent mixtures or a suitable solvent cyclised through closing the triazinone ring on functionalised imidazolidines in both cases. All fused azaisocytosine-containing congeners were investigated with the purpose of preselecting possible drug candidates with a better selectivity that could be suitable for further more detailed drug development studies. The majority of test molecules revealed strong antiproliferative effects in most tumour cell cultures and they were more cytotoxic against tumour cells than anticancer drug - pemetrexed. These cytotoxicities may be associated with the activation of initiator and executioner caspases (confirmed for compound 12) which are inducers of apoptosis. Simultaneously, three bioisosteres bearing the trifluoromethyl moiety at the C-3 and the ortho substitution at the phenyl ring (10, 12 and 13) proved to be the most promising in terms of selectivity as they were less or equally toxic to normal cells as pemetrexed. It was shown that isosteric replacement of the ethyl group in antitumour active congeners by the trifluoromethyl or isopropyl group was favourable for the selectivity of the designed drug-like molecules. Almost all new compounds revealed the protective effects in an ex vivo model of oxidatively stressed rat erythrocytes (better or comparable than that of ascorbic acid/Trolox), proving that they are safe to red blood cells. The statistically significant and predictive QSAR equations were derived that describe relationships between some pharmacokinetic descriptors (such as log Ka, HSA, fu, brain, Caco-2, log Kp) and lipophilicity parameters of test molecules. Among all molecules with anticancer profile, the possible drug candidates seem to be 10, 12, 13, 19 and 21 which are the least toxic for normal cells, deprived of haemolytic effects on oxidatively-stressed red blood cells and have the optimum pharmacokinetic descriptors in terms of their lipophilicity parameters. Because of a high development potential they should be utilised in further more extended in vivo investigations aimed at developing novel less toxic anticancer agents.

Nucleobase synthesis in interstellar ices.

The synthesis of nucleobases in natural environments, especially in interstellar molecular clouds, is the focus of a long-standing debate regarding prebiotic chemical evolution. Here we report the simultaneous detection of all three pyrimidine (cytosine, uracil and thymine) and three purine nucleobases (adenine, xanthine and hypoxanthine) in interstellar ice analogues composed of simple molecules including H2O, CO, NH3 and CH3OH after exposure to ultraviolet photons followed by thermal processes, that is, in conditions that simulate the chemical processes accompanying star formation from molecular clouds. Photolysis of primitive gas molecules at 10 K might be one of the key steps in the production of nucleobases. The present results strongly suggest that the evolution from molecular clouds to stars and planets provides a suitable environment for nucleobase synthesis in space.

Crystal Structure of Mistletoe Lectin I (ML-I) from Viscum album in Complex with 4-N-Furfurylcytosine at 2.85 Å Resolution.

BACKGROUND: Viscum album (the European mistletoe) is a semi-parasitic plant, which is of high medical interest. It is widely found in Europe, Asia, and North America. It contains at least three distinct lectins (i.e. ML-I, II, and III), varying in molecular mass and specificity. Among them, ML-I is in focus of medical research for various activities, including anti-cancer activities. To understand the molecular basis for such medical applications, a few studies have already addressed the structural and functional analysis of ML-I in complex with ligands. In continuation of these efforts, we are reporting the crystal structure of ML from Viscum album in complex with the nucleic acid oxidation product 4-N-furfurylcytosine (FC) refined to 2.85 Å resolution. FC is known to be involved in different metabolic pathways related to oxidative stress and DNA modification. METHODS: X-ray suitable hexagonal crystals of the ML-I/FC complex were grown within four days at 294 K using the hanging drop vapor diffusion method. Diffraction data were collected up to a resolution of 2.85 Å. The ligand affinity was verified by in-silico docking. RESULTS: The high-resolution structure was refined subsequently to analyze particularly the active site conformation and a binding epitope of 4-N-furfurylcytosine. A distinct 2Fo-Fc electron density at the active site was interpreted as a single FC molecule. The specific binding of FC is achieved also through hydrophobic interactions involving Tyr76A, Tyr115A, Glu165A, and Leu157A of the ML-I A-chain. The binding energy of FC to the active site of ML-I was calculated as well to be -6.03 kcal mol-1. CONCLUSION: In comparison to other reported ML-I complexes, we observed distinct differences in the vicinity of the nucleic acid base binding site upon interaction with FC. Therefore, data obtained will provide new insights in understanding the specificity, inhibition, and cytotoxicity of the ML-I A-chain, and related RIPs.

Binding investigation and preliminary optimisation of the 3-amino-1,2,4-triazin-5(2H)-one core for the development of new Fyn inhibitors.

Fyn tyrosine kinase inhibitors are considered potential therapeutic agents for a variety of human cancers. Furthermore, the involvement of Fyn kinase in signalling pathways that lead to severe pathologies, such as Alzheimer's and Parkinson's diseases, has also been demonstrated. In this study, starting from 3-(benzo[d][1,3]dioxol-5-ylamino)-6-methyl-1,2,4-triazin-5(2H)-one (VS6), a hit compound that showed a micromolar inhibition of Fyn (IC50 = 4.8 µM), we computationally investigated the binding interactions of the 3-amino-1,2,4-triazin-5(2H)-one scaffold and started a preliminary hit to lead optimisation. This analysis led us to confirm the hypothesised binding mode of VS6 and to identify a new derivative that is about 6-fold more active than VS6 (compound 3, IC50 = 0.76 µM).

Synthesis and Biological Characterization of Aryl Uracil Inhibitors of Hepatitis C Virus NS5B Polymerase: Discovery of ABT-072, a trans-Stilbene Analog with Good Oral Bioavailability.

ABT-072 is a non-nucleoside HCV NS5B polymerase inhibitor that was discovered as part of a program to identify new direct-acting antivirals (DAAs) for the treatment of HCV infection. This compound was identified during a medicinal chemistry effort to improve on an original lead, inhibitor 1, which we described in a previous publication. Replacement of the amide linkage in 1 with a trans-olefin resulted in improved compound permeability and solubility and provided much better pharmacokinetic properties in preclinical species. Replacement of the dihydrouracil in 1 with an N-linked uracil provided better potency in the genotype 1 replicon assay. Results from phase 1 clinical studies supported once-daily oral dosing with ABT-072 in HCV infected patients. A phase 2 clinical study that combined ABT-072 with the HCV protease inhibitor ABT-450 provided a sustained virologic response at 24 weeks after dosing (SVR24) in 10 of 11 patients who received treatment.

Reaction of S-geranyl-2-thiouracil modified oligonucleotides with alkyl amines leads to the N2-alkyl isocytosine derivatives.

S-Geranylated 2-thiouridines (geS2Us) are unique hydrophobic modified nucleosides identified very recently in bacterial tRNAs. Our study on the synthesis of geS2Ura-containing oligonucleotides (geS2U-RNA and geS2dU-DNA) revealed a fast substitution of the S-geranyl moiety by methylamine (frequently used in oligonucleotide deprotection procedures) or n-butylamine, providing the corresponding N2-alkyl isocytosine (R2isoCyt) derivatives. To retain the S-geranyl moiety in the DNA or RNA chains, the optimized deprotection protocol with 8 M ethanolic ammonia should be applied. The oligomers bearing the R2isoCyt heterocycle (R2isoC-RNA and R2isodC-DNA) are less hydrophobic than the corresponding S2U- and geS2U-modified oligomers, whereas, contrary to the previously reported data, geS2dU-DNA and geS2U-RNA exhibit significantly higher lipophilicity than the parent S2Ura-containing oligonucleotides. Thermodynamic studies revealed that: (a) both geS2Ura- and R2isoCyt-modified oligomers exhibit similar hybridization properties towards DNA and RNA templates, and (b) the R2isoCyt nucleobase preferentially hybridizes to guanine moiety in the DNA/DNA and RNA/RNA duplexes.

Antimetabolites: A First Synthesis of a New Class of Cytosine Thioglycoside Analogs.

A first synthesis of a new class of novel cytosine thioglycoside analogs from readily available starting materials has been described. The key step of this protocol is the formation of sodium pyrimidine-4-thiolate via condensation of N'-arylidene-2-cyanoacetohydrazides with sodium cyanocarbonimidodithioate salt, followed by coupling with halo sugars to give the corresponding cytosine thioglycoside analogs. Ammonolysis of the latter compounds afforded the free thioglycosides.

Direct conjugation of peptides and 5-hydroxymethylcytosine in DNA.

Recent discovery of functional 5-hydroxymethylcytosine in vertebrate genomes prompted for elaboration of methods to localize this modification at the nucleotide resolution level. Among several covalent modification-based approaches, atypical activity of cytosine-5 DNA methyltransferases to couple small molecules to 5-hydroxymethylcytosine stands out for acceptance of broad range of ligands. We went further to explore the possibility for methyltransferase-maintained coupling of compounds possessing autonomous functions. Functionalization of DNA was achieved by direct conjugation of chemically synthesized peptides of regular structure. Sequence, residue, and position-specific coupling of DNA containing 5-hydroxymethylcytosine and different peptides has been demonstrated, with the nature of the resulting conjugates confirmed by protease treatment and mass spectrometry. Coupling products were compatible with affinity-driven separation from the unmodified DNA. This approach highlights an emerging avenue toward the enzymatic, sequence-specific DNA functionalization, enabling a single step merge of the DNA and peptide moieties into a bifunctional entity.

Synthesis and complementary self-association of novel lipophilic π-conjugated nucleoside oligomers.

A series of lipophilic nucleosides comprising natural and non-natural bases that are π-conjugated to a short oligophenylene-ethynylene fragment has been synthesized. These bases comprise guanosine, isoguanosine, and 2-aminoadenosine as purine heterocycles, and cytidine, isocytosine and uridine as complementary pyrimidine bases. The hydrogen-bonding dimerization and association processes between complementary bases were also studied by (1)H NMR and absorption spectroscopy in order to obtain the relevant association constants.

A general approach to the synthesis of 5-S-functionalized pyrimidine nucleosides and their analogues.

A general and efficient approach was developed for the introduction of S-functionality at the C-5 position of cytosine and uracil nucleosides and their analogues. The key step is a palladium-catalyzed C-S coupling of the corresponding 5-bromo nucleoside derivative and alkyl thiol. The butyl 3-mercaptopropionate coupling products were further converted to the corresponding disulphides, the stable precursors of 5-mercaptopyrimidine nucleosides.

Biomimetic polyorganosiloxanes: model compounds for new materials.

The chemistry of N-organosilylalkyl-substituted heterocyclic bases (thymine, adenine and cytosine) is described, covering the structures of model compounds, the synthesis of substituted oligo-siloxanes and a preliminary report of the synthesis of a poly(organosiloxane) with pendant N-alkyl(heterocycle) functionalities. N-Alkenylthymines CH2=CH(CH2)(n)T (T = thymine, n = 1 (1), 2 (2), 3 (3)) have been prepared and 2 hydrosilylated to form PhMe2Si(CH2)4T (5). Alternatively, 5 was prepared by reaction of PhMe2Si(CH2)4Br (6) with (O,O-SiMe3)2T, a method which has also been used to prepare PhMe2Si(CH2)4A (7) and PhMe2Si(CH2)4C (8) (A = adenine, C = cytosine). Model di- and tri-siloxanes [Br(CH2)4(Me)2Si]2O (10), Me3SiOSi(Me)2(CH2)4Br (11), PhMe2SiOSi(Me)2(CH2)4Br (12) and (Me3SiO)2(Me)Si(CH2)4Br (13) have been prepared by hydrosilylation of H2C[double bond, length as m-dash]C(H)(CH2)4Br with an appropriate hydrosiloxane and used to prepare Me3SiO(Me)2Si(CH2)4T (14), Me3SiO(Me)2Si(CH2)4A (15) (both from 11), and (Me3SiO)2(Me)Si(CH2)4T (16), (Me3SiO)2(Me)Si(CH2)4A (17) (both from 13). 10 reacts with thymine to give a mixture of the pyrimidocyclophane cyclo-T-N,N-[(CH2)4(Me)2Si]2O (19) and [T(CH2)4Si(Me)2]2O (20), while cytosine reacts similarly to form cyclo-C-N,N-[(CH2)4(Me)2Si]2O (21; as an imine) and [C(CH2)4Si(Me)2]2O (22); adenine only generates [A(CH2)4Si(Me)2]2O (18) in an analogous synthesis. Using a related protocol, polymeric {[MeSi(O)(CH2)4Br]2[Me2SiO]98}n (23) has been converted to {[MeSi(O)(CH2)4T]2[Me2SiO]98}n (24) and {[MeSi(O)(CH2)4A]2[Me2SiO]98}n (25). The structures of 4, 5, 8, 19 and 21, along with a 2 : 1 adduct of 5 with Ni(dithiobiuret)2 (9) are reported.

N(4)-Acyl derivatives as lipophilic prodrugs of cidofovir and its 5-azacytosine analogue, (S)-HPMP-5-azaC: chemistry and antiviral activity.

Even number fatty acid residues-docosanoyl (behenoyl) and stearoyl were selected for introduction to the N(4)-position of (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (HPMPC, cidofovir), and its 5-azacytosine counterpart, (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (HPMP-5-azaC) with the aim to prepare a new type of lipophilic prodrugs. The study on the influence of these modifications to the stability and biological activity of both antivirals was performed. Different reactivity of both systems towards acylation reactions was also found: the 4-NH2 group of cidofovir was more reactive compared to that of HPMP-5-azaC. In 5-azacytosine derivatives, we found mostly a destabilizing effect of the N(4)-acylation but this could be compensated by a positive influence of the esterification of the phosphonate group. Chemical stability of the 5-azacytosine moiety in the HPMP series is increasing in the following order: HPMP-5-azaC

Chromone-fused cytosine analogues: synthesis, biological activity, and structure-activity relationship.

The preparation of a series of novel chromone-fused cytosine analogues, i.e., chromeno[2,3-d]pyrimidines has been carried out from substituted 2-amino-4-oxo-4H-chromene-3-carbonitriles with urea, thiourea, and guanidine under different reaction conditions. These chromone-fused cytosine analogues were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv strain and different microbial pathogenic strains in cell culture for their structure-activity relationships, respectively. Among the synthesized compounds, 2d, 3a, and 4e showed better results against Mycobacterium tuberculosis H37Rv. The compounds 2a, 2b, and 3a showed potential antibacterial activity against E. coli and P. aeruginosa, while the majority of compounds were found to be active against S. aureus as compared to ampicillin. The synthesized cytosine analogues having an imine (-C&dbnd;NH) have been less sensitive to the bacterial and fungal strains but have a more beneficial effect on Mycobacterium tuberculosis H37Rv.

Pyrimidine-fused heterocyclic frameworks based on an N4-arylcytosine scaffold: synthesis, characterization, and PNA oligomerization of the fluorescent cytosine analogue 5,6-benzopC.

A synthesis of an intrinsically fluorescent cytosine analogue 5,6-benzopC has been developed utilizing the reductive Ni-mediated cyclization of an N4-aryl,N4-(Boc)cytosine intermediate as a key step. 5,6-BenzopC was found to possess interesting fluorescence properties (Φ = 0.79, EtOH; Stoke's shift 113 nm). Peptide nucleic acid (PNA) oligomerization of the 5,6-benzopC monomer was carried out, followed by hybridization studies with complementary deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which showed the modification to be well tolerated in the sequence contexts examined. Initial attempts to synthesize the heterocyclic skeleton present in 5,6-benzopC resulted in the discovery of routes to the pyrimido[1,6-a]benzimidazole, pyrimido[1,6-a]quinazoline, and pyrimido[1,6-a]benzo[b]6-bora-1,3-diazine heterocyclic frameworks.

Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring.

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.

Convenient and efficient syntheses of N(6)- and N(4)- substituted adenines and cytosines and their 2'-deoxyribosides.

Convenient and efficient methods of the synthesis of N(6)- and N(4)-substituted derivatives of adenine and cytosine and their 2'-deoxyribosides were developed. The reactions of either unprotected nucleobases (adenine, cytosine) or unprotected 2'-deoxyribosides with aryl or alkyl aldehydes give corresponding Schiff bases that can be reduced to the target title compounds with high overall yields. In the case of aryl aldehydes the imine derivatives are obtained in the presence of methoxides in methanol and reduced with sodium borohydride. The corresponding reactions with alkyl aldehydes require the use of acetic acid and borane dimethyl sulfide complex instead.

Isocytosine-based inhibitors of xanthine oxidase: design, synthesis, SAR, PK and in vivo efficacy in rat model of hyperuricemia.

Structure-activity relationship studies were carried out for lead generation following structure-guided design approach from an isocytosine scaffold identified earlier for xanthine oxidase inhibition. A 470-fold improvement in in vitro IC(50) was obtained in the process. Five most potent compounds with nanomolar IC(50) values were selected for pharmacokinetics and in vivo experiments. The best compound showed good in vivo activity when administered intraperitoneally but was not active by oral route. The results suggest that improvement in oral exposure could improve the in vivo efficacy of this series.

New cytosine derivatives as inhibitors of DNA methylation.

DNA cytosine methylation catalyzed by DNA methyltransferase 1 (DNMT1) is an epigenetic method of gene expression regulation and development. Changes in methylation pattern lead to carcinogenesis. Inhibition of DNMT1 activity could be a good strategy of safe and efficient epigenetic therapy. In this work, we present a novel group of cytosine analogs as inhibitors of DNA methylation. We show new methods of synthesis and their effect on in vitro reaction of DNA methylation. Almost all of analyzed compounds inhibit DNA methyltransferase activity in the competitive manner. K(i) values for the most potent compound 4-N-furfuryl-5,6-dihydroazacytosines is 0.7 µM. These compounds cause also a decrease of 5-methylcytosine (m(5)C) level in DNA of mammalian HeLa and HEK293 cells.

Differential effect of three base modifications on DNA thermostability revealed by high resolution melting.

High resolution melting (HRM) can detect and quantify the presence of 5-methylcytosine (5mC) in DNA samples, but the ability of HRM to diagnose other DNA modifications remains unexplored. The DNA bases N6-methyladenine and 5-hydroxymethylcytosine occur across almost all phyla. While their function remains controversial, their presence perturbs DNA structure. Such modifications could affect gene regulation, chromatin condensation and DNA packaging. Here, we reveal that DNA containing N6-methyladenine or 5-hydroxymethylcytosine exhibits reduced thermal stability compared to cytosine-methylated DNA. These thermostability changes are sufficiently divergent to allow detection and quantification by HRM analysis. Thus, we report that HRM distinguishes between sequence-identical DNA differing only in the modification type of one base. This approach is also able to distinguish between two DNA fragments carrying both N6-methyladenine and 5-methylcytosine but differing only in the distance separating the modified bases. This finding provides scope for the development of new methods to characterize DNA chemically and to allow for low cost screening of mutant populations of genes involved in base modification. More fundamentally, contrast between the thermostabilizing effects of 5mC on dsDNA compared with the destabilizing effects of N6-methyladenine (m6A) and 5-hydroxymethylcytosine (5hmC) raises the intriguing possibility of an antagonistic relationship between modification types with functional significance.