Design and synthesis of uracil/thiouracil based quinoline scaffolds as topoisomerases I/II inhibitors for chemotherapy: A new hybrid navigator with DFT calculation.

In this work, a novel promising hybrid mode of uracil/thiouracil based quinoline pharmacophore i.e. 5a-f was rationalized and synthesized based on rigidification and lipophilic principles, and following the reported pharmacophoric features of camptothecin & doxorubicin. Concurrently, a non-rigid mode pharmacophore i.e. 7a-f was also designed and synthesized. The anti-proliferative activity of the compounds was assessed against three different cancer cell lines, namely A549 lung cancer, MCF-7 breast adenocarcinoma, and HepG-2 hepatic carcinoma. Further, promising candidates were evaluated against A549, and MCF-7 and for their ability to inhibit topoisomerases I &II. Compound 5f was observed to be the most active congener, displaying the highest cell inhibition of 84.4% for topoisomerase I and 92%, for topoisomerase II at a concentration of 100 µM. When its cytotoxicity was evaluated against A549 cells, 5f arrested the cell cycle at the S phase and increased the apoptosis ratio by 46.31%. DFT calculation of 5f showed higher dipole moment and greater negative energy values (-247531.510 kcal/mol) with positive & negative poles, and better stability reflection. Furthermore, molecular docking of 5f to both enzymes showed good agreement with the biological assessment. This study has given insight for further consideration of the highly promising hybrid 5f.

Novel benzenesulfonamide-thiouracil conjugates with a flexible N-ethyl acetamide linker as selective CA IX and CA XII inhibitors.

Novel benzenesulfonamide derivatives linked to diverse functionalized thiouracils through a flexible N-ethyl acetamide linker were designed and synthesized as carbonic anhydrase (CA) inhibitors. The synthesized candidates demonstrated a potent inhibitory activity against four different CA isoforms in the nanomolar range. Compound 10d showed more than twofold higher potency than the reference AAZ against CA II with Ki of 5.65 and 12 nM, respectively. Moreover, compounds 10d and 20 revealed potent activity against CA IX with Ki of 18.1 and 14.2 nM, respectively. In addition, 10c, 10d, 11b, 11c, and 20 demonstrated high potency against the CA XII isozyme with a Ki range of 4.18-4.8 nM. Most of the synthesized derivatives displayed preferential selectivity toward the CA IX and CA XII isoforms over CA I and CA II. Compounds 11a and 20 exhibited favorable selectivity toward CA IX over CA II with a selectivity index (SI) of 14.36 and 16.62, respectively, and toward CA XII over CA II with SI of 71.01 and 51.19, respectively. Molecular docking simulations showed that the synthesized conjugates adopted comparable binding modes in the CA I, CA II, CA IX, and CA XII isoforms, involving the deep fitting of the sulfonamide moiety in the base of the CA active site via chelation of the Zn2+ ion and H-bond interaction with the key amino acids Thr199 and/or Thr200. Moreover, the N-ethyl acetamide flexible linker enables the substituted thiouracils and fused thiouracil tail to achieve multiple interactions with the surrounding hydrophobic and hydrophilic regions.

The Effect of Methylation on the Triplet-State Dynamics of 2-Thiouracil: Time-Resolved Photoelectron Spectroscopy of 2-Thiothymine.

The ultrafast internal conversion and intersystem crossing dynamics of 2-thiouracil (2TU) and 2-thiothymine (2TT) are studied using time-resolved photoelectron spectroscopy to investigate the effect of methylation on the deactivation mechanism. Like other thiobases, the triplet manifold is populated with high quantum yields via the lowest singlet excited state, which is dark in absorption. This study focuses on the lowest triplet state and the role of two minima, with sulfur-out-of-plane and slightly boat-like geometries, in the intersystem crossing dynamics back to the ground state.

Electron attachment dynamics following UV excitation of iodide-2-thiouracil complexes.

The dynamics of low energy electron attachment to the thio-substituted uracil analog 2-thiouracil are investigated using time-resolved photoelectron spectroscopy (TRPES) of iodide-2-thiouracil (I-·2TU) binary clusters. In these experiments, the anions are excited at pump energies of 4.16 and 4.73 eV, and the ensuing dynamics are probed by photodetachment at 1.59 and 3.18 eV. Upon excitation near the vertical detachment energy (4.16 eV), dipole bound (DB) and valence bound (VB) anion signals appear almost instantaneously, and the DB state of the 2TU anion undergoes an ultrafast decay (∼50 fs). At 4.73 eV, there is no evidence for a DB state, but features attributed to two VB states are seen. The transient negative ions formed by photoexcitation decay by autodetachment and I- fragmentation. The I- dissociation rates and their dependence on excitation energy agree reasonably well with the Rice-Ramsperger-Kassel-Marcus calculations. Notable differences with respect to TRPES of the related iodide-uracil anion are observed and discussed.

A Computational QSAR, Molecular Docking and In Vitro Cytotoxicity Study of Novel Thiouracil-Based Drugs with Anticancer Activity against Human-DNA Topoisomerase II.

Computational chemistry, molecular docking, and drug design approaches, combined with the biochemical evaluation of the antitumor activity of selected derivatives of the thiouracil-based dihydroindeno pyrido pyrimidines against topoisomerase I and II. The IC50 of other cell lines including the normal human lung cell line W138, lung cancer cell line, A549, breast cancer cell line, MCF-7, cervical cancer, HeLa, and liver cancer cell line HepG2 was evaluated using biochemical methods. The global reactivity descriptors and physicochemical parameters were computed, showing good agreement with the Lipinski and Veber's rules of the drug criteria. The molecular docking study of the ligands with the topoisomerase protein provides the binding sites, binding energies, and deactivation constant for the inhibition pocket. Various biochemical methods were used to evaluate the IC50 of the cell lines. The QSAR model was developed for colorectal cell line HCT as a case study. Four QSAR statistical models were predicted between the IC50 of the colorectal cell line HCT to correlate the anticancer activity and the computed physicochemical and quantum chemical global reactivity descriptors. The predictive power of the models indicates a good correlation between the observed and the predicted activity.

Development of 1-(4-(Substituted)piperazin-1-yl)-2-((2-((4-methoxybenzyl)thio)pyrimidin-4-yl)oxy)ethanones That Target Poly (ADP-Ribose) Polymerase in Human Breast Cancer Cells.

A number of uracil amides cleave poly (ADP-ribose) polymerase and therefore novel thiouracil amide compounds were synthesized and screened for the loss of cell viability in a human-estrogen-receptor-positive breast cancer cell line. The synthesized compounds exhibited moderate to significant efficacy against human breast cancer cells, where the compound 5e IC50 value was found to be 18 µM. Thouracil amide compounds 5a and 5e inhibited the catalytical activity of PARP1, enhanced cleavage of PARP1, enhanced phosphorylation of H2AX, and increased CASPASE 3/7 activity. Finally, in silico analysis demonstrated that compound 5e interacted with PARP1. Hence, specific thiouracil amides may serve as new drug-seeds for the development of PARP inhibitors for use in oncology.

Design, synthesis, ADME, biological evaluation and molecular dynamic studies of natural and synthetic remedy of Herpes simplex virus type-1.

Garlic (known as; Allium sativum) is one of the most widely used medicinal plants in the world. Allicin is the major agent of garlic that gives its known pharmacological activities as anti-inflammatory, antibacterial, antifungal, antiviral and antioxidant agent. It could be extracted from bulbs of Allium sativum by water extraction to give allicin in low yield therefore other better methods were followed for extraction such as ultrasonic-assisted method that gives good yield. Attempts to optimize allicin extraction were found with sliced garlic at 25 °C for 90 minute of extraction for maximum yield (112µg/mL). Allicin was subjected to its evaluation as anti-herpetic against herpes simplex virus 1 (HSV-1) and exhibited a promising activity compared to acyclovir which was used as a reference standard. On the other hand, a novel synthetic amantadine derivative was evaluated as antiherpetic agent and prepared from the reaction of 2-thiouracil-5-sulphonyl chloride with amantadine hydrochloride in pyridine. The synergestic effect of allicin and the amantadine derivative was evaluated against HSV-1, using both in silico molecular docking as for dynamics simulations. Thymidine kinase target enzyme was chosen to analyze any possible interactions, as well as any protein-ligand stability. Furthermore, some of properties of the potential HSV-1 thymidine kinase target inhibitor of the amantadine derivative were analyzed.

SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting.

Cell type-specific transcriptome analysis is an essential tool for understanding biological processes in which diverse types of cells are involved. Although cell isolation methods such as fluorescence-activated cell sorting (FACS) in combination with transcriptome analysis have widely been used so far, their time-consuming and harsh procedures limit their applications. Here, we report a novel in vivo metabolic RNA sequencing method, SLAM-ITseq, which metabolically labels RNA with 4-thiouracil in a specific cell type in vivo followed by detection through an RNA-seq-based method that specifically distinguishes the thiolated uridine by base conversion. This method has successfully identified the cell type-specific transcriptome in three different tissues: endothelial cells in brain, epithelial cells in intestine and adipocytes in white adipose tissue. As this method does not require isolation of cells or RNA prior to the transcriptomic analysis, SLAM-ITseq provides an easy yet accurate snapshot of the transcriptional state in vivo.

Investigating the mapping of chromophore excitations onto the electron detachment spectrum: photodissociation spectroscopy of iodide ion-thiouracil clusters.

Laser photodissociation spectroscopy (3.1-5.7 eV) has been applied to iodide complexes of the non-native nucleobases, 2-thiouracil (2-TU), 4-thiouracil (4-TU) and 2,4-thiouracil (2,4-TU), to probe the excited states and intracluster electron transfer as a function of sulphur atom substitution. Photodepletion is strong for all clusters (I-·2-TU, I-·4-TU and I-·2,4-TU) and is dominated by electron detachment processes. For I-·4-TU and I-·2,4-TU, photodecay is accompanied by formation of the respective molecular anions, 4-TU- and 2,4-TU-, behaviour that is not found for other nucleobases. Notably, the I-·2TU complex does not fragment with formation of its molecular anion. We attribute the novel formation of 4-TU- and 2,4-TU- to the fact that these valence anions are significantly more stable than 2-TU-. We observe further similar behaviour for I-·4-TU and I-·2,4-TU relating to the general profile of their photodepletion spectra, since both strongly resemble the intrinsic absorption spectra of the respective uncomplexed thiouracil molecule. This indicates that the nucleobase chromophore excitations are determining the clusters' spectral profile. In contrast, the I-·2-TU photodepletion spectrum is dominated by the electron detachment profile, with the near-threshold dipole-bound excited state being the only distinct spectral feature. We discuss these observations in the context of differences in the dipole moments of the thionucleobases, and their impact on the coupling of nucleobase-centred transitions onto the electron detachment spectrum.

Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA.

Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.

Synthesis of Novel 2-Thiouracil-5-Sulfonamide Derivatives as Potent Inducers of Cell Cycle Arrest and CDK2A Inhibition Supported by Molecular Docking.

In an effort to discover potent anticancer agents, 2-thiouracil-5-sulfonamides derivatives were designed and synthesized. The cytotoxic activity of all synthesized compounds was investigated against four human cancer cell lines viz A-2780 (ovarian), HT-29 (colon), MCF-7 (breast), and HepG2 (liver). Compounds 6b,d-g, and 7b showed promising anticancer activity and significant inhibition of CDK2A. Moreover, they were all safe when tested on WI38 normal cells with high selectivity index for cancer cells. Flow cytometric analysis for the most active compound 6e displayed induction of cell growth arrest at G1/S phase (A-2780 cells), S phase (HT-29 and MCF-7 cells), and G2/M phase (HepG2 cells) and stimulated the apoptotic death of all cancer cells. Moreover, 6e was able to cause cycle arrest indirectly through enhanced expression of cell cycle inhibitors p21 and p27. Finally, molecular docking of compound 6e endorsed its proper binding to CDK2A, which clarifies its potent anticancer activity.

Core-Level Spectroscopy of 2-Thiouracil at the Sulfur L1- and L2,3-Edges Utilizing a SASE Free-Electron Laser.

In this paper, we report X-ray absorption and core-level electron spectra of the nucleobase derivative 2-thiouracil at the sulfur L1- and L2,3-edges. We used soft X-rays from the free-electron laser FLASH2 for the excitation of isolated molecules and dispersed the outgoing electrons with a magnetic bottle spectrometer. We identified photoelectrons from the 2p core orbital, accompanied by an electron correlation satellite, as well as resonant and non-resonant Coster-Kronig and Auger-Meitner emission at the L1- and L2,3-edges, respectively. We used the electron yield to construct X-ray absorption spectra at the two edges. The experimental data obtained are put in the context of the literature currently available on sulfur core-level and 2-thiouracil spectroscopy.

Defining the RNA interactome by total RNA-associated protein purification.

The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)-associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA-associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA-protein complexes using silica beads. In a variant approach (PAR-TRAPP), RNAs were labelled with 4-thiouracil prior to 350 nm crosslinking. PAR-TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR-TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA-peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization.

Global analysis of RNA metabolism using bio-orthogonal labeling coupled with next-generation RNA sequencing.

Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.

Different Oxidation Pathways of 2-Selenouracil and 2-Thiouracil, Natural Components of Transfer RNA.

Sulfur- and selenium-modified uridines present in the wobble position of transfer RNAs (tRNAs) play an important role in the precise reading of genetic information and tuning of protein biosynthesis in all three domains of life. Both sulfur and selenium chalcogens functionally operate as key elements of biological molecules involved in the protection of cells against oxidative damage. In this work, 2-thiouracil (S2Ura) and 2-selenouracil (Se2Ura) were treated with hydrogen peroxide at 1:0.5, 1:1, and 1:10 molar ratios and at selected pH values ranging from 5 to 8. It was found that Se2Ura was more prone to oxidation than its sulfur analog, and if reacted with H2O2 at a 1:1 or lower molar ratio, it predominantly produced diselenide Ura-Se-Se-Ura, which spontaneously transformed to a previously unknown Se-containing two-ring compound. Its deselenation furnished the major reaction product, a structure not related to any known biological species. Under the same conditions, only a small amount of S2Ura was oxidized to form Ura-SO2H and uracil (Ura). In contrast, 10-fold excess hydrogen peroxide converted Se2Ura and S2Ura into corresponding Ura-SeOnH and Ura-SOnH intermediates, which decomposed with the release of selenium and sulfur oxide(s) to yield Ura as either a predominant or exclusive product, respectively. Our results confirmed significantly different oxidation pathways of 2-selenouracil and 2-thiouracil.

Observation of Enhanced Dissociative Photochemistry in the Non-Native Nucleobase 2-Thiouracil.

We present the first study to measure the dissociative photochemistry of 2-thiouracil (2-TU), an important nucleobase analogue with applications in molecular biology and pharmacology. Laser photodissociation spectroscopy is applied to the deprotonated and protonated forms of 2-TU, which are produced in the gas-phase using electrospray ionization mass spectrometry. Our results show that the deprotonated form of 2-thiouracil ([2-TU-H]-) decays predominantly by electron ejection and hence concomitant production of the [2-TU-H]· free-radical species, following photoexcitation across the UVA-UVC region. Thiocyanate (SCN-) and a m/z 93 fragment ion are also observed as photodecay products of [2-TU-H]- but at very low intensities. Photoexcitation of protonated 2-thiouracil ([2-TU·H]+) across the same UVA-UVC spectral region produces the m/z 96 cationic fragment as the major photofragment. This ion corresponds to ejection of an HS· radical from the precursor ion and is determined to be a product of direct excited state decay. Fragment ions associated with decay of the hot ground state (i.e., the ions we would expect to observe if 2-thiouracil was behaving like UV-dissipating uracil) are observed as much more minor products. This behaviour is consistent with enhanced intersystem crossing to triplet excited states compared to internal conversion back to the ground state. These are the first experiments to probe the effect of protonation/deprotonation on thionucleobase photochemistry, and hence explore the effect of pH at a molecular level on their photophysical properties.

Selective 4-Thiouracil Labeling of RNA Transcripts within Latently Infected Cells after Infection with Human Cytomegalovirus Expressing Functional Uracil Phosphoribosyltransferase.

Infections with human cytomegalovirus (HCMV) are highly prevalent in the general population as the virus has evolved the capacity to undergo distinct replication strategies resulting in lytic, persistent, and latent infections. During the latent life cycle, HCMV resides in subsets of cells within the hematopoietic cell compartment, including hematopoietic progenitor cells (HPCs) and peripheral blood monocytes. Since only a small fraction of these cell types harbor viral genomes during natural latency, identification and analysis of distinct changes mediated by viral infection are difficult to assess. In order to characterize latent infections of HPCs, we used an approach that involves complementation of deficiencies within the human pyrimidine salvage pathway, thus allowing for conversion of labeled uracil into rUTP. Here, we report the development of a recombinant HCMV that complements the defective human pyrimidine salvage pathway, allowing incorporation of thiol containing UTP into all RNA species that are synthesized within an infected cell. This virus grows to wild-type kinetics and can establish a latent infection within two distinct culture models of HCMV latency. Using this recombinant HCMV, we report the specific labeling of transcripts only within infected cells. These transcripts reveal a transcriptional landscape during HCMV latency that is distinct from uninfected cells. The utility of this labeling system allows for the identification of distinct changes within host transcripts and will shed light on characterizing how HCMV establishes and maintains latency.IMPORTANCE HCMV is a significant pathogen that accounts for a substantial amount of complications within the immunosuppressed and immunocompromised. Of particular significance is the capacity of HCMV to reactivate within solid tissue and bone marrow transplant recipients. While it is known that HCMV latency resides within a fraction of HPCs and monocytes, the exact subset of cells that harbor latent viral genomes during natural infections remain uncharacterized. The capacity to identify changes within the host transcriptome during latent infections is critical for developing approaches that therapeutically or physically eliminate latent viral genome containing cells and will represent a major breakthrough for reducing complications due to HCMV reactivation posttransplant. In this report, we describe the generation and use of a recombinant HCMV that allows specific and distinct labeling of RNA species that are produced within virally infected cells. This is a critical first step in identifying how HCMV affects the host cell during latency and more importantly, allows one to characterize cells that harbor latent HCMV.

Biomolecules of 2-Thiouracil, 4-Thiouracil and 2,4-Dithiouracil: A DFT Study of the Hydration, Molecular Docking and Effect in DNA:RNAMicrohelixes.

The molecular structure of 2-thiouracil, 4-thiouracil and 2,4-dithiouracil was analyzed under the effect of the first and second hydration shell by using the B3LYP density functional (DFT) method, and the results were compared to those obtained for the uracil molecule. A slight difference in the water distribution appears in these molecules. On the hydration of these molecules several trends in bond lengths and atomic charges were established. The ring in uracil molecule appears easier to be deformed and adapted to different environments as compared to that when it is thio-substituted. Molecular docking calculations of 2-thiouracil against three different pathogens: Bacillus subtilis, Escherichia coli and Candida albicans were carried out. Docking calculations of 2,4-dithiouracil ligand with various targeted proteins were also performed. Different DNA: RNA hybrid microhelixes with uridine, 2-thiouridine, 4-thiouridine and 2,4-dithiouridine nucleosides were optimized in a simple model with three nucleotide base pairs. Two main types of microhelixes were analyzed in detail depending on the intramolecular H-bond of the 2'-OH group. The weaker Watson-Crick (WC) base pair formed with thio-substituted uracil than with unsubstituted ones slightly deforms the helical and backbone parameters, especially with 2,4-dithiouridine. However, the thio-substitution significantly increases the dipole moment of the A-type microhelixes, as well as the rise and propeller twist parameters.

Radiation Induced One-Electron Oxidation of 2-Thiouracil in Aqueous Solutions.

Oxidative damage to 2-thiouracil (2-TU) by hydroxyl (•OH) and azide (●N3) radicals produces various primary reactive intermediates. Their optical absorption spectra and kinetic characteristics were studied by pulse radiolysis with UV-vis spectrophotometric and conductivity detection and by time-dependent density functional theory (TD-DFT) method. The transient absorption spectra recorded in the reactions of •OH with 2-TU depend on the concentration of 2-TU, however, only slightly on pH. At low concentrations, they are characterized by a broad absorption band with a weakly pronounced maxima located at λ = 325, 340 and 385 nm, whereas for high concentrations, they are dominated by an absorption band with λmax ≈ 425 nm. Based on calculations using TD-DFT method, the transient absorption spectra at low concentration of 2-TU were assigned to the ●OH-adducts to the double bond at C5 and C6 carbon atoms (3●, 4●) and 2c-3e bonded ●OH adduct to sulfur atom (1…●OH) and at high concentration of 2-TU also to the dimeric 2c-3e S-S-bonded radical in neutral form (2●). The dimeric radical (2●) is formed in the reaction of thiyl-type radical (6●) with 2-TU and both radicals are in an equilibrium with Keq = 4.2 × 103 M-1. Similar equilibrium (with Keq = 4.3 × 103 M-1) was found for pH above the pKa of 2-TU which involves admittedly the same radical (6●) but with the dimeric 2c-3e S-S bonded radical in anionic form (2●-). In turn, ●N3-induced oxidation of 2-TU occurs via radical cation with maximum spin location on the sulfur atom which subsequently undergoes deprotonation at N1 atom leading again to thiyl-type radical (6●). This radical is a direct precursor of dimeric radical (2●).

Regioselectivity of thiouracil alkylation: Application to optimization of Darapladib synthesis.

Darapladib is one of the most potent Lp-PLA2 (Lipoprotein-associated phospholipase A2) inhibitor with an IC50 of 0.25 nM. We demonstrate that a crucial step of Darapladib synthesis was not correctly described in the literature, leading to the production of wrong regioisomers. Moreover we show that the inhibitory activity is directly linked to the position on N1 since compounds bearing alkylation on different sites have potentially less interaction within the active site of Lp-PLA2.