Insights into Cisplatin Binding to Uracil and Thiouracils from IRMPD Spectroscopy and Tandem Mass Spectrometry.

The monofunctional primary complexes cis-[PtCl(NH3)2(L)]+, formed by the reaction of cisplatin, a major chemotherapeutic agent, with four nucleobases L, i.e., uracil (U), 2-thiouracil (2SU), 4-thiouracil (4SU), and 2,4-dithiouracil (24dSU), have been studied by a combination of infrared multiple photon dissociation (IRMPD) action spectroscopy in both the fingerprint (900-1900 cm-1) and the N-H/O-H stretching (3000-3800 cm-1) ranges, energy-resolved collision-induced dissociation (CID) mass spectrometry, and density functional calculations at the B3LYP/LACVP/6-311G** level. On the basis of the comparison across the experimental features and the linear IR spectra of conceivable structures, the cisplatin residue is found to promote a monodentate interaction preferentially with the O4(S4) atoms of the canonical forms of U, 4SU, and 24dSU and to the S2 atom of 2SU, yielding the most stable structures. Additional absorptions reveal the presence of minor, alternative tautomers in the sampled ion populations of 2SU and 24dSU, underlying the ability of cisplatin to increase the prospect of (therapeutically beneficial) nucleic acid strand disorder. Implication of these evidence may provide insights into drug mechanism and design.

Ru(II) complexes containing uracil nucleobase analogs with cytotoxicity against tumor cells.

We report on chemistry and cytotoxic studies of four new ruthenium (II) complexes containing uracil derivatives. All compounds are neutral, presenting the formula [Ru(PPh3)2(2TU)2] (1), [Ru(PPh3)2(6m2TU)2] (2), [Ru(dppb)(2TU)2] (3) and [Ru(dppb)(6m2TU)2] (4), where PPh3 = triphenylphosphine; dppb = 1,4-bis(diphenylphosphino)butane, 2TU = 2-thiouracil and 6m2TU = 6-methyl-2-thiouracil. They were characterized using NMR, UV-vis and IR spectroscopies, microanalytical analysis and mass spectrometry. Furthermore, the crystal structures of 1-4 were determined by single-crystal X-ray diffraction. The coordination of 2-thiouracil derivatives with ruthenium increases regions able to carry out hydrogen bonds with the biological targets, such as DNA. We evaluated the interaction of the complexes with DNA by UV/Vis spectrophotometric titration, and as a result, the values of DNA-binding constants are in the range of 0.8-1.8 × 104 M-1. Moreover, the interaction of the complexes with BSA was investigated. In vitro, activities against B16-F10 (mouse melanoma), HepG2 (human hepatocellular carcinoma), HL-60 (human promyelocytic leukemia) and K562 (human chronic myelocytic leukemia) and non-tumor cells: PBMC (human peripheral blood mononuclear cells activated with concanavalin A - human lymphoblast) were carried out. Cytotoxicity assays revealed that complexes (2) and (4) present biological activity against tumor cells comparable with oxaliplatin, the reference platinum drug, revealing that they are promising molecules for developing new antitumor compounds.

A gene encoding a DUF523 domain protein is involved in the conversion of 2-thiouracil into uracil.

Modified nucleotides are present in many RNA species in all Domains of Life. While the biosynthetic pathways of such nucleotides are well studied, much less is known about the degradation of RNAs and the return to the metabolism of modified nucleotides, their respective nucleosides or heterocyclic bases. Using an E. coli uracil auxotroph, we screened the metagenomic libraries for genes, which would allow the conversion of 2-thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. We show that a gene encoding a protein consisting of previously uncharacterized Domain of Unknown Function 523 (DUF523) is responsible for such phenotype. We have purified this recombinant protein and demonstrated that it contains a FeS cluster. The substitution of cysteines, which have been predicted to form such clusters, with alanines abolished the growth phenotype. We conclude that DUF523 is involved in the conversion of 2-thiouracil into uracil in vivo.

Synthesis, characterization and molecular docking studies of thiouracil derivatives as potent thymidylate synthase inhibitors and potential anticancer agents.

Thymidylate synthase (TS), one of folate-dependent enzymes, is a key and well-recognized target for anticancer agents. In this study, a series of 6-aryl-5-cyano thiouracil derivatives were designed and synthesized in accordance with essential pharmacophoric features of known TS inhibitors. Nineteen compounds were screened in vitro for their anti-proliferative activities toward HePG-2, MCF-7, HCT-116, and PC-3 cell lines. Compounds [Formula: see text], [Formula: see text], and 24 exhibited high anti-proliferative activity, comparable to that of 5-fluorouracil. Additionally, ten compounds with potent anti-proliferative activities were further evaluated for their ability to inhibit TS enzyme. Six compounds ([Formula: see text], [Formula: see text], [Formula: see text], 22, 23 and 24) demonstrated potent dose-related TS inhibition with [Formula: see text] values ranging from 1.57 to [Formula: see text]. The in vitro TS activity results were consistent with those of the cytotoxicity assay where the most potent anti-proliferative compounds of the series showed good TS inhibitory activity comparable to that of 5-fluorouracil. Furthermore, molecular docking studies were carried out to investigate the binding pattern of the designed compounds with the prospective target, TS (PDB-code: 1JU6).

Hybrids of thienopyrimidinones and thiouracils as anti-tubercular agents: SAR and docking studies.

A number of hybrid molecules containing thienopyrimidinones and thiouracil moieties were designed, synthesized and tested against Mycobacterium tuberculosis H37Ra wherein it was observed that the compounds 11-14 exhibited antitubercular activity in vitro (MIC 7.6-19.1 µg/mL, 12-35 µM) against dormant stage while the compound 15 exhibited antitubercular activity in vitro against dormant (MIC 23.4 µg/mL, 41 µM) as well as active (MIC 25.4 µg/mL, 45 µM) stage. Structural modifications of the compound 15 were carried out to study the structure-activity relationship and it was observed that the compound 18 exhibited antitubercular activity comparable to the compound 15. Cytotoxicity studies revealed that these molecules were non-toxic. The docking study of the compound 15 showed that there was binding with the active site of mycobacterial pantothenate synthetase. Further docking studies led to the synthesis of the compounds 16 and 17 and the antitubercular activity screening results showed that these compounds have significant antitubercular activity. The compounds 15-18 (MIC 11-29 µg/mL, 19-51 µM) can be used as starting points for further optimization. The synthetic strategies used in the present work have potential to prepare a large number of compounds for further refinement of structures and the present results will be very useful in the development of a new class of antimycobacterial agents.

Design, synthesis and antimicrobial activities of thiouracil derivatives containing triazolo-thiadiazole as SecA inhibitors.

A series of novel thiouracil derivatives containing a triazolo-thiadiazole moiety (7a-7l) have been synthesized by structural modifications on a lead SecA inhibitor, 2. All the compounds have been evaluated for their antibacterial activities against Bacillus amyloliquefaciens, Staphylococcus aureus, and Bacillus subtilis. Compounds 7d and 7g were also tested for their inhibitory activities against SecA ATPase due to their promising antimicrobial activities. The inhibitory activity of compound 7d was found to be higher than that of 2. Molecular docking work suggests that compound 7d might bind at a pocket close to the ATPase ATP-binding domain.

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.

In silico/in vitro study of hybrid D-modified steroidal alkylator anticancer activity using uridine phosphorylase as target protein.

BACKGROUND: In order to reduce toxicity and to enhance anticancer activity of nitrogen mustards, three hybrid steroidal esters were synthesized and tested in vitro against human pancreatic cancer cells expressing uridine phosphorylase (UPase). The inhibition potency against a target protein implicated in the chemotherapy of solid tumors, such as UPase, is of fundamental importance in the design and synthesis of new anticancer drugs. MATERIALS AND METHODS: MTT colorimetric assay and molecular docking were employed for the in vitro and in silico drug evaluation, respectively. RESULTS: A difference in cell sensitivity was found, which followed the known different UPase expression in the cell lines. Molecular docking studies on UPase protein, revealed the tested compounds to be bound to the binding cavity of the protein, with different affinity. Between the two D-modified compounds, the D-homo-aza (lactam)-hybrid compound (C2) was found to interact with the protein in a more efficient way. CONCLUSION: The molecular docking data were in accordance with the in vitro results, where the lactam steroid alkylator showed significantly higher cytostatic and cytotoxic activity than the non-D-modified compounds, which also correlated with the level of UPase expression in the pancreatic cancer cells.

Human hair follicle pigmentary unit as a direct target for modulators of melanogenesis, as studied by [14C]-2-thiouracil incorporation.

The purpose of this study was to evaluate human hair follicle melanogenic activity using the [14C]-2-thiouracil, which was known to incorporate into nascent melanins. Results obtained on pigmented, grey and non-pigmented hair follicles demonstrated that [(14)C]-2-TU incorporation was restricted to the melanogenic compartment with a strong accumulation located around dermal papilla and within the fibre of pigmented hair follicles. Quantitative analysis of [(14)C]-2-TU incorporation showed a significant increase in pigmented hair follicles upon stimulation with 1 microm forskolin concomitant to an increase in tyrosinase levels. A strong significant decrease in [14C]-2-TU incorporation was noted, when hair follicles were incubated with the tyrosinase competitive inhibitor kojic acid (200 microm). Incubation with the MC1-R agonist alpha-MSH (0.2 microm) did not induce a significant stimulation of hair melanogenesis. The present model could thus represent a useful new tool to identify modulators of human hair pigmentation.

Mechanism of selective incorporation of the melanoma seeker 2-thiouracil into growing melanin.

The mechanism of selective incorporation of 2-thiouracil (TU), a highly specific melanoma seeker, into growing melanins was investigated both in vitro and in vivo. Methods used included direct analysis of the melanins, by evaluation of the absorption at 350 nm (A350) and chemical degradation coupled with HPLC quantitation of pigment makers, i.e., pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), as well as biosynthetic experiments involving tyrosinase-catalyzed oxidation of DOPA, 5,6-dihydroxyindole (DHI), and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Injection of radiolabeled TU into melanoma-bearing mice resulted in a rapid incorporation of the drug into the tumor pigment, with a substantial decrease in A350 and in PTCA yields. Similar changes in the absorption properties were observed in biosynthetic melanins prepared in the presence of TU, whereas the yields of PTCA and PDCA varied depending on the pigment precursor used. When incubated with DOPA in the presence of tyrosinase, TU profoundly modified the normal course of melanogenesis, favoring formation of a complex mixture of addition products consisting mainly of 6-S-thiouracil-DOPA as well as DHI-TU adducts. The latter were obtained in larger amounts by enzymatic oxidation of DHI in the presence of TU and were identified as the 3- and 2-substituted adducts 1 and 2, the dimer 3, and the trimer 4. Similar reactions carried out on DHICA yielded the 4-substituted adduct 5, the dimer 6, and the trimer 7. A new mechanistic scheme for the incorporation of TU into growing melanin is proposed, which envisages nucleophilic attack of the thioureylene moiety of TU to transient quinonoid intermediates in the melanin pathway, chiefly dopaquinone and 5,6-indolequinones, followed by entrainment of the resulting adducts into the growing pigment via oxidative copolymerization with DHICA and/or DHI.

Signalling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes.

To understand the signalling mechanisms involved in the dual stimulatory effects of endothelin-1 (ET-1) on DNA synthesis and melanization in cultured human melanocytes, we analysed the biological profile of ET-1 receptor and determined the effects of ET-1 on the protein kinase C, cyclic AMP system and mitogen-activated protein kinase (MAP kinase) in comparison with their relevant stimulants. The photoaffinity labelling of ET-1 receptors with Denny-Jaff reagents revealed an ET-1 receptor with a molecular mass of 51 kDa in human melanocytes. The ET(A) receptor subtype-sensitive antagonist BQ123(50 nM) or pertussis toxin (100 ng/ml) significantly suppressed the ET-1-induced intracellular calcium mobilization, indicating the presence of pertussis toxin-sensitive G-protein-coupled ET(A) receptors. An assay of protein kinase C activity revealed that 10nM ET-1 translocated cytosolic protein kinase C to membrane-bound protein kinase C within 5 min of the start of incubation. In contrast, receptor-mediated melanocyte activation by ET-1 was accompanied by an elevated level of cyclic AMP (4-fold over control) after 10-60 min of incubation, whereas 60 min of incubation of human melanocytes with c-Kit or c-Met ligands such as stem cell factor (10 nM) or basic fibroblast growth factor (10 nM) did not elevate the cyclic AMP level. We have also demonstrated that a specific tyrosine kinase inhibitor, tyrphostin B-42 (10 microM), inhibited the ET-1-induced growth stimulation, suggesting the involvement of the tyrosine kinase pathway in growth stimulation. Consistently, an assay of MAP kinase revealed that ET-1 caused a 10-fold activation of MAP kinase after 5 min of incubation with human melanocytes in a similar way to tyrosine kinase ligands such as stem cell factor and hepatocyte growth factor. Further, the DNA synthesis stimulated by the c-Kit ligand stem cell factor at a concentration of 1 nM was synergistically enhanced by 5 nM ET-1. These results suggest that ET-induced dual cellular events in human melanocytes are closely associated with cross-talk between the protein kinase C and A and tyrosine kinase pathways.

Specific incorporation of 2-thiouracil into biological melanins.

2-Thiouracil (TU), an antithyroid drug, is generally recognized as a highly specific melanoma seeker owing to its capability of being selectively accumulated into active melanin-producing tissues. We recently reported evidence that in vitro TU is capable of reacting with dopaquinone (DQ), an early intermediate in melanin biosynthesis, to give an addition product characterized as 6-S-thiouracildopa (TD). However, several aspects of the mechanism of the uptake of TU into melanin in vivo still need to be clarified. We report here the extremely rapid incorporation of [2-14C]thiouracil into melanoma tumors growing subcutaneously in mice and show its selective accumulation into melanin by isolation and purification of the pigment fraction. Formation of the TD adduct in the tumor was examined by HPLC analysis of the soluble fractions of the tissue homogenates: however, no trace of TD could be detected on account of its rapid oxidation by the melanogenic enzyme tyrosinase, as evidenced by in vitro kinetic measurements. Monitoring the course of the tyrosinase-catalyzed oxidation of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in the presence of TU, at various molar ratios, provided evidence for the ability of the drug to affect melanogenesis by interaction with biosynthetic intermediates beyond the DQ stage, suggesting other possible modes for its chemical binding to the growing pigment.

Synthetic approaches to a carboranyl thiouracil.

Thiouracil is selectively incorporated into melanotic murine melanomas during melanin synthesis. This selectivity makes thiouracil a likely vehicle for boron in the diagnosis and therapy of melanoma. Several synthetic routes to thiouracils bearing an alkyl decacarboranyl group attached to various positions on the ring have been investigated. The successful syntheses of three new alkynyl thiouracils and the conversion of one of them into a carboranyl thiouracil are described.

Selective incorporation of thiouracil into murine metastatic melanomas.

The uptake and retention of 14C-thiouracil and 125I-thiouracil in small lung metastases of B16 murine melanoma was studied in beige mice injected intravenously with melanoma cells. By impulse counting of excised tumor and organ pieces, a high concentration of radioactivity was found in the lung metastases, as compared to normal tissues. The highest tumor/organ concentration ratios appeared 24 h after injection of the radiolabeled thiouracil. A separate autoradiographic study on the disposition of 14C-thiouracil in mice with melanoma metastases confirmed the impulse counting results and also showed the absence of any other site of retention of radioactivity except for hair follicles and to some extent the thyroid. The selective uptake of 14C- and 125I-thiouracil in melanomas depends on their acceptance as false melanin precursors, making them specific markers for growing melanin. The results indicate that radiolabeled thiouracil may be useful for clinical diagnosis and, possibly, therapy of malignant melanotic melanomas.

Accumulation of chlorpromazine and thiouracil by human melanoma cells in culture.

The uptake (total radioactivity in intact cells) and incorporation (radioactivity bound to acid-precipitable material) of 14C-chlorpromazine (CPZ) and 14C-thiouracil (TU) were studied using a library of 3 human fibroblast strains and 13 tumour cell lines. In contrast to previous studies using rodent melanomas in vivo, the melanoma lines, including lines with high tyrosinase and melanin contents, did not take up more CPZ and TU than non-melanoma cells (fibroblasts, HeLa cells). Incorporation of CPZ was also broadly similar in all cell types studied. TU was selectively incorporated into the melanoma line having a high tyrosinase and melanin content but not into lines with high tyrosinase activity and low melanin content. While supporting the possibility of selective therapy for heavily-pigmented melanomas using labelled TU derivatives, these results suggest that the action of potentially melanoma-affined compounds should be further evaluated in human cells. Unlabelled CPZ or TU was not selectively toxic to melanoma cells. Unexpectedly, methylation-sensitive tumour cells (Mer-phenotype) were highly resistant to TU, thus providing a new experimental tool for understanding the genesis of this phenotype in vivo.

Radio-iodine-labelled 5-iodo-2-thiouracil: a potential radiopharmaceutical for establishing the viability of ocular melanoma after radiation therapy.

Radio-iodine-labelled thiouracil has been evaluated as a radiopharmaceutical for establishing the viability of ocular melanoma after radiation treatment. The uptake of 125I-5-iodo-2-thiouracil (125I-ITU) was studied in X-ray irradiated and non-irradiated melanotic melanomas implanted in Syrian golden hamsters. Uptake of 125I-ITU in melanomas 4 days after irradiation with 40 Gy X-ray was 25% of the value found in non-irradiated controls, 12 days after such treatment it was 10% of that value. Twenty-one days after radiation treatment the melanomas showed regrowth and uptake of 125I-ITU was about equal to that in non-treated controls. Uptake of 125I-ITU in melanomas after 10 Gy X-ray irradiation was higher and uptake in tumours after 20 Gy was only slightly lower than the uptake by non-irradiated melanomas. The results indicate that the iodine labelled-thiouracil uptake test may be useful as an additional diagnostic issue for assessing the viability of ocular melanoma after radiation therapy.

Iodothiouracil as a melanoma localizing agent.

Thiouracil and various derivatives are selectively incorporated into the melanin pigment of melanomas during biosynthesis by serving as false melanin precursors. Using the transplantable Harding-Passey melanoma carried in BALB/c mice, we have extended our previous studies with sulfur-35 (35S) thiouracil. The persistence of high levels of [35S]thiouracil in tumor for periods of up to 2 wk has been demonstrated; during this time the drug content in normal tissues returned to near background levels. The variety of iodine isotopes available makes iodothiouracil a particularly promising melanoma-localizing agent. Tumor uptake and biodistribution of [35S]thiouracil and iodothiouracil (both iodine-127 (127I) and iodine-125 (125I) labeled) have been compared and were found to be essentially the same. The selectivity of [125I]thiouracil for melanoma has been qualitatively demonstrated by autoradiography of whole-body sections and quantitated by analysis of tumor and selected tissues. Iodothiouracil was also shown to localize in remote secondary metastases using a metastatic variant of the Harding-Passey melanoma currently being developed in our laboratory. These studies confirm the melanoma localizing capabilities of an iodinated thiouracil, and therefore the potential of using iodinated thiouracil derivatives for diagnosis and therapy of melanotic melanomas.

Uptake of 123I-5-iodo-2-thiouracil, a possible radiopharmaceutical for noninvasive detection of ocular melanoma, in melanotic and amelanotic melanomas in hamsters.

The uptake of 123I-5-iodo-2-thiouracil in melanotic and amelanotic melanoma implanted in Syrian golden hamsters was studied. A selective accumulation was found in the tumours. Uptake of 123ITU in melanotic melanomas was 4 to 5 times the uptake in amelanotic ones. For both tumours high ratios of tumours versus non-tumour were found. The high accumulation of 123ITU in both kinds of tumours and the high tumour versus non-tumour ratios suggest that 123ITU may be a promising radiopharmaceutical for the detection of ocular melanoma.

Effects of topical retinoic acid on intracutaneously implanted S91 melanoma in mice.

Previous studies have demonstrated that retinoids possess antineoplastic properties against melanoma. The purpose of this study was to determine whether topically applied retinoic acid could prevent melanoma development in syngeneic mice after intracutaneous cell inoculation. Trans-retinoic acid in DMSO was applied daily for 28 days after melanoma implantation and tumor growth was quantitated by the uptake of [14C]thiouracil, a tracer compound specific for melanoma which is incorporated linearly according to the weight of the tumor. Marked reduction in tumor growth was noted at the highest concentration (0.1%) tested and lesser but significantly decreased tumor growth patterns were also realized at lower concentrations in a dose-dependent manner. Thus, topically applied retinoic acid is capable of inhibiting S91 melanoma growth in vivo.

Detection of melanomas. Approach with radiolabeled false precursors of melanin synthesis.

Thiouracil is a thiol-containing pyrimidine that is selectively incorporated into cells that synthesize melanin. In an effort to delineate further the specificity and dynamics of uptake, we injected thiouracil labeled with radioactive carbon into S91 melanoma-bearing mice; biopsy specimens were taken of the tumors and organs at various time intervals thereafter. The data showed a substantial uptake of thiouracil by the melanomas, with peak uptake occurring at 24 hours. All other organs examined showed only minor amounts of radioactivity, which probably reflected the presence of thiouracil in the blood perfusing these tissues. Because of its incorporation into melanomas, the use of radioactive thiouracil has potential as a marker for tumor growth, as a diagnostic tracer compound, and as a carrier for chemotherapeutic agents.