Repurposing of Antifungal Drug Flucytosine/Flucytosine Cocrystals for Anticancer Activity against Prostate Cancer Targeting Apoptosis and Inflammatory Signaling Pathways.

This study aimed to repurpose the antifungal drug flucytosine (FCN) for anticancer activity together with cocrystals of nutraceutical coformers sinapic acid (SNP) and syringic acid (SYA). The cocrystal screening experiments with SNP resulted in three cocrystal hydrate forms in which two are polymorphs, namely, FCN-SNP F-I and FCN-SNP F-II, and the third one with different stoichiometry in the asymmetric unit (1:2:1 ratio of FCN:SNP:H2O, FCN-SNP F-III). Cocrystallization with SYA resulted in two hydrated cocrystal polymorphs, namely, FCN-SYA F-I and FCN-SYA F-II. All the cocrystal polymorphs were obtained concomitantly during the slow evaporation method, and one of the polymorphs of each system was produced in bulk by the slurry method. The interaction energy and lattice energies of all cocrystal polymorphs were established using solid-state DFT calculations, and the outcomes correlated with the experimental results. Further, the in vitro cytotoxic activity of the cocrystals was determined against DU145 prostate cancer and the results showed that the FCN-based cocrystals (FCN-SNP F-III and FCN-SYA F-I) have excellent growth inhibitory activity at lower concentrations compared with parent FCN molecules. The prepared cocrystals induce apoptosis by generating oxidative stress and causing nuclear damage in prostate cancer cells. The Western blot analysis also depicted that the cocrystals downregulate the inflammatory markers such as NLRP3 and caspase-1 and upregulate the intrinsic apoptosis signaling pathway marker proteins, such as Bax, p53, and caspase-3. These findings suggest that the antifungal drug FCN can be repurposed for anticancer activity.

Exploring Product Release from Yeast Cytosine Deaminase with Metadynamics.

The yeast cytosine deaminase (yCD) enzyme/5-fluorocytosine prodrug system is a promising candidate for targeted chemotherapeutics. After conversion of the prodrug into the toxic chemotherapeutic drug, 5-fluorouracil (5-FU), the slow product release from the enzyme limits the overall catalytic efficiency of the enzyme/prodrug system. Here, we present a computational study of the product release of the anticancer drug, 5-FU, from yCD using metadynamics. We present a comparison of the 5-FU drug to the natural enzyme product, uracil. We use volume-based metadynamics to compute the free energy landscape for product release and show a modest binding affinity for the product to the enzyme, consistent with experiments. Next, we use infrequent metadynamics to estimate the unbiased release rate from Kramers time-dependent rate theory and find a favorable comparison to experiment with a slower rate of product release for the 5-FU system. Our work demonstrates how adaptive sampling methods can be used to study the protein-ligand unbinding process for engineering enzyme/prodrug systems and gives insights into the molecular mechanism of product release for the yCD/5-FU system.

Click Nucleic Acid Mediated Loading of Prodrug Activating Enzymes in PEG-PLGA Nanoparticles for Combination Chemotherapy.

The simultaneous delivery of multiple therapeutics to a single site has shown promise for cancer targeting and treatment. However, because of the inherent differences in charge and size between drugs and biomolecules, new approaches are required for colocalization of unlike components in one delivery vehicle. In this work, we demonstrate that triblock copolymers containing click nucleic acids (CNAs) can be used to simultaneously load a prodrug enzyme (cytosine deaminase, CodA) and a chemotherapy drug (doxorubicin, DOX) in a single polymer nanoparticle. CNAs are synthetic analogs of DNA comprised of a thiolene backbone and nucleotide bases that can hybridize to complementary strands of DNA. In this study, CodA was appended with complementary DNA sequences and fluorescent dyes to allow its encapsulation in PEG-CNA-PLGA nanoparticles. The DNA-modified CodA was found to retain its enzyme activity for converting prodrug 5-fluorocytosine (5-FC) to active 5-fluorouracil (5-FU) using a modified fluorescent assay. The DNA-conjugated CodA was then loaded into the PEG-CNA-PLGA nanoparticles and tested for cell cytotoxicity in the presence of the 5-FC prodrug. To study the effect of coloading DOX and CodA within a single nanoparticle, cell toxicity assays were run to compare dually loaded nanoparticles with nanoparticles loaded only with either DOX or CodA. We show that the highest level of cell death occurred when both DOX and CodA were simultaneously entrapped and delivered to cells in the presence of 5-FC.

Molecular Analyses Support the Safety and Activity of Retroviral Replicating Vector Toca 511 in Patients.

Purpose: Toca 511 is a gammaretroviral replicating vector encoding cytosine deaminase that selectively infects tumor cells and converts the antifungal drug 5-fluorocytosine into the antineoplastic drug 5-fluorouracil, which directly kills tumor cells and stimulates antitumor immune responses. As part of clinical monitoring of phase I clinical trials in recurrent high-grade glioma, we have performed extensive molecular analyses of patient specimens to track vector fate.Patients and Methods: Toca 511 and Toca FC (extended-release 5-fluorocytosine) have been administered to 127 high-grade glioma patients across three phase I studies. We measured Toca 511 RNA and DNA levels in available body fluids and tumor samples from patients to assess tumor specificity. We mapped Toca 511 integration sites and sequenced integrated Toca 511 genomes from patient samples with detectable virus. We measured Toca 511 levels in a diverse set of tissue samples from one patient.Results: Integrated Toca 511 is commonly detected in tumor samples and is only transiently detected in blood in a small fraction of patients. There was no believable evidence for clonal expansion of cells with integrated Toca 511 DNA, or preferential retrieval of integration sites near oncogenes. Toca 511 sequence profiles suggest most mutations are caused by APOBEC cytidine deaminases acting during reverse transcription. Tissue samples from a single whole-body autopsy affirm Toca 511 tumor selectivity.Conclusions: Toca 511 and Toca FC treatment was not associated with inappropriate integration sites and clonal expansion. The vector is tumor-selective and persistent in patients who received Toca 511 injections. Clin Cancer Res; 24(19); 4680-93. ©2018 AACR.

Insight into the recognition mechanism of DNA cytosine-5 methyltransferases (DNMTs) by incorporation of acyclic 5-fluorocytosine (FC) nucleosides into DNA.

DNA cytosine-5 methyltransferase (DNMT) catalyzes methylation at the C5 position of cytosine in the CpG sequence in double stranded DNA to give 5-methylCpG (mCpG) in the epigenetic regulation step in human cells. The entire reaction mechanism of DNMT is divided into six steps, which are scanning, recognition, flipping, loop locking, methylation, and releasing. The methylation and releasing mechanism are well-investigated; however, few reports are known about other reaction steps. To obtain insight into the reaction mechanism, we planned the incorporation of acyclic nucleosides, which make it easy to flip out the target nucleobase, into oligodeoxynucleotides (ODNs) and investigated the interaction between the ODN and DNMT. Here, we describe the design and synthesis of ODNs containing new acyclic 5-fluorocytosine nucleosides and their physiological and biological properties, including their interactions with DNMT. We found that the ODNs containing the acyclic 5-fluorocytosine nucleoside showed higher flexibility than those that contain 5-fluoro-2'-deoxycytidine. The observed flexibility of ODNs is expected to influence the scanning and recognition steps due to the decrease in helicity of the B-form.

Novel potentially antifungal hybrids of 5-flucytosine and fluconazole: Design, synthesis and bioactive evaluation.

A series of novel potentially antifungal hybrids of 5-flucytosine and fluconazole were designed, synthesized and characterized by 1H NMR, 13C NMR, IR and HRMS spectra. Bioactive assay manifested that some prepared compounds showed moderate to good antifungal activities in comparison with fluconazole and 5-flucytosine. Remarkably, the 3,4-dichlorobenzyl hybrid 7h could inhibit the growth of C. albicans ATCC 90023 and clinical resistant strain C. albicans with MIC values of 0.008 and 0.02 mM, respectively. The active molecule 7h could not only rapidly kill C. albicans but also efficiently permeate membrane of C. albicans. Molecular docking study revealed that compound 7h could interact with the active site of CACYP51 through hydrogen bond. Quantum chemical studies were also performed to explain the high antifungal activity. Further preliminary mechanism research suggested that molecule 7h could intercalate into calf thymus DNA to form a steady supramolecular complex, which might block DNA replication to exert the powerful bioactivities.

Flucytosine analogues obtained through Biginelli reaction as efficient combinative antifungal agents.

Invasive fungal infection is a problem that continues to challenge the healthcare sector. New antifungals and new therapeutic strategies are needed to address this challenge. We previously reported that the combination of a synthetic compound with a drug with known mechanism of action is a good strategy to treat aggressive and resistant fungi. Here we revisited our approach and synthesized structural analogues of flucytosine, which is a synthetic antifungal and is being studied for its use in combination therapy with other antifungal drugs. Pyrimidin-one and -thione (often known as DHPM's) as flucytosine analogues were obtained through a Biginelli reaction of corresponding aldehydes, ethylacetoacetate and urea/thiourea. Structure was confirmed by FTIR, 1HNMR, 13CNMR, COSY and MS (ESI+) analysis. All the newly synthesized derivatives were evaluated for the antifungal activity alone and in combination of two most commonly used antifungal drugs, amphotericin B and fluconazole against different clinically isolated Candida albicans strains. Minimum inhibitory concentration results confirmed that BG4 possess high antifungal activity against all the tested strains (MIC = 1-32 µg/ml). For all the combinations with amphotericin B and fluconazole, 37% were synergistic followed by 30% additive and 24% indifferent interactions. Interestingly, 9% antagonistic interaction was observed when BG1 and BG3 were combined with fluconazole, however, no antagonistic interaction was observed with amphotericin B. In-depth studies of all the synergies were done by constructing isobolograms with nine different ratio combinations. These results warrant the use of DHPM derivatives as chemosensitising agents which could lower down the dosages of the antifungal drugs to treat invasive fungal diseases.

Cleavage of DNA containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases.

A systematic study of the cleavage of DNA sequences containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases (REs) was performed and the results compared with the same sequences containing natural pyrimidine bases, uracil or 5-methylcytosine. The results show that some REs recognize fluorine as a hydrogen on cytosine and cleave the corresponding sequences where the presence of m5dC leads to blocking of the cleavage. However, on uracil, the same REs recognize the F as a methyl surrogate and cleave the sequences which are not cleaved if uracil is incorporated instead of thymine. These results are interesting for understanding the recognition of DNA sequences by REs and for manipulation of the specific DNA cutting.

Bioactive Core-Shell Nanofiber Hybrid Scaffold for Efficient Suicide Gene Transfection and Subsequent Time Resolved Delivery of Prodrug for Anticancer Therapy.

Nanofiber scaffold's ability to foster seemingly nonexistent interface with the cells enables them to effectively deliver various bioactive molecules to cells in the vicinity. Among such bioactive molecules, therapeutically active nucleic acid has been the most common candidate. In spite of such magnanimous efforts in this field, it remains a paradox that suicide gene delivery by nanofibers has never been sought for anticancer application. To investigate such a possibility, in the present work, a composite core-shell nanofiberous scaffold has been realized which could efficiently transfect suicide gene into cancer cells and simultaneously deliver prodrug, 5-Fluorocytosine (5-FC) in a controlled and sustained manner. The scaffold's ability to instigate apoptosis by suicide gene therapy in nonsmall lung cancer cells (A549) was ascertained at both phenotypic and genotypic levels. A cascade of events starting from suicide gene polyplex release from nanofibers, transfection, and expression of cytosine deaminase-uracil phosphoribosyltransferase (CD::UPRT) suicide gene by A549; subsequent prodrug release; and its metabolic conversion into toxic intermediates which finally culminates in host cells apoptosis has been monitored in a time-dependent manner. This work opens up new application avenues for nanofiber-based scaffolds which can effectively manage cancer prognosis.

Quantum chemical investigations on the nonradiative deactivation pathways of cytosine derivatives.

The nonradiative deactivation pathways of cytosine derivatives (cytosine, 5-fluorocytosine, 5-methylcytosine, and 1-methycytosine) and their tautomers are investigated by quantum chemical calculations, and the substituent effects on the deactivation process are examined. The MS-CASPT2 method is employed in the excited-state geometry optimization and also in the search for conical intersection points, and the potential energy profiles connecting the Franck-Condon point, excited-state minimum energy structures, and the conical intersection points are investigated. Our calculated vertical and adiabatic excitation energies are in quite good agreement with the experimental results, and the relative barrier heights leading to the conical intersections are correlated with the experimentally observed excite-state lifetimes, where the calculated barrier heights are in the order of cytosine < 5-methylcytosine < 5-fluorocytosine.

Versatile types of polysaccharide-based supramolecular polycation/pDNA nanoplexes for gene delivery.

Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple ß-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations with adamantane-functionalized polysaccharides is an effective strategy for the production of new nanoplex delivery systems.

Excited-state structure, vibrations, and nonradiative relaxation of jet-cooled 5-fluorocytosine.

The S0 → S1 vibronic spectrum and S1 state nonradiative relaxation of jet-cooled keto-amino 5-fluorocytosine (5FCyt) are investigated by two-color resonant two-photon ionization spectroscopy at 0.3 and 0.05 cm(­1) resolution. The 0(0)(0) rotational band contour is polarized in-plane, implying that the electronic transition is (1)ππ*. The electronic transition dipole moment orientation and the changes of rotational constants agree closely with the SCS-CC2 calculated values for the (1)ππ* (S1) transition of 5FCyt. The spectral region from 0 to 300 cm(­1) is dominated by overtone and combination bands of the out-of-plane ν1' (boat), ν2' (butterfly), and ν3' (HN­C6H twist) vibrations, implying that the pyrimidinone frame is distorted out-of-plane by the (1)ππ* excitation, in agreement with SCS-CC2 calculations. The number of vibronic bands rises strongly around +350 cm(­1); this is attributed to the (1)ππ* state barrier to planarity that corresponds to the central maximum of the double-minimum out-of-plane vibrational potentials along the ν1', ν2', and ν3' coordinates, which gives rise to a high density of vibronic excitations. At +1200 cm(­1), rapid nonradiative relaxation (k(nr) ≥ 10(12) s(­1)) sets in, which we interpret as the height of the (1)ππ* state barrier in front of the lowest S1/S0 conical intersection. This barrier in 5FCyt is 3 times higher than that in cytosine. The lifetimes of the ν' = 0, 2ν1', 2ν2', 2ν1' + 2ν2', 4ν2', and 2ν1' + 4ν2' levels are determined from Lorentzian widths fitted to the rotational band contours and are τ ≥ 75 ps for ν' = 0, decreasing to τ ≥ 55 ps at the 2ν1' + 4ν2' level at +234 cm(­1). These gas-phase lifetimes are twice those of S1 state cytosine and 10­100 times those of the other canonical nucleobases in the gas phase. On the other hand, the 5FCyt gas-phase lifetime is close to the 73 ps lifetime in room-temperature solvents. This lack of dependence on temperature and on the surrounding medium implies that the 5FCyt nonradiative relaxation from its S1 ((1)ππ*) state is essentially controlled by the same ~1200 cm(­1) barrier and conical intersection both in the gas phase and in solution.

Near-IR-induced, UV-induced, and spontaneous isomerizations in 5-methylcytosine and 5-fluorocytosine.

Monomeric 5-methylcytosine (5mCyt) and 5-fluorocytosine (5FCyt) were studied using the matrix-isolation method. In 5mCyt and 5FCyt, the most stable form, dominating in low-temperature matrixes, is the amino-hydroxy (AH) tautomer. For both compounds, irradiation of the matrixes with near-IR laser light or with broadband near-IR or mid-IR light induces interconversions between the two rotamers of tautomer AH. In addition, for matrixes kept in darkness, a spontaneous tunneling conversion of the higher-energy hydroxy conformer (with the OH group directed toward the N3 atom) into the lower-energy form (OH directed toward N1) was occurring, with half-life time of 70 min for 5mCyt and 127 min for 5FCyt. These tunneling processes are much faster than that found for unsubstituted cytosine, where the half-life time is more than 30 h. UV irradiation of 5mCyt (at 316 nm) led to phototautomeric conversion of the amino-oxo form into the amino-hydroxy tautomer. Another phototransformation induced by irradiation of 5mCyt at 316 nm was the cleavage of the C-N bond in the amino-oxo form, resulting in generation of the open-ring conjugated isocyanate product. Irradiation of 5mCyt at shorter waves (λ ≤ 310 nm) induced the syn-anti photoisomerization within the imino-oxo forms of the compound. For matrix-isolated 5FCyt, the amount of the amino-oxo form was very small (with respect to the amino-hydroxy tautomer), while the imino-oxo isomers were not detected at all.

Enabling tablet product development of 5-fluorocytosine through integrated crystal and particle engineering.

The antifungal drug, 5-fluorocytosine (FC), is marketed as a capsule (250 or 500 mg strength) instead of the preferred tablet dosage form. Through systematic characterization of solid-state properties, including mechanical properties, we identify tabletability and poor physical stability of FC as the problems that likely have prevented the successful development of a FC tablet product. We then design an FC oxalate 2:1 salt (FCOXA21), based on established relationship between crystal structure and properties, to address these deficient properties. FCOXA21 is subsequently used to develop a direct compression tablet product using predictive and material-sparing powder characterization tools, that is, ring shear cell for powder flowability and compaction simulator for powder tabletability. The initial tablet formulation, which contains 84.5% (wt %) FCOXA21, exhibits excellent tabletability but inadequate flowability. We solve the powder flowability problem through controlling the particle size of FCOXA21. A batch of FCOXA21 tablets (500 mg FC equivalent dose) is then prepared. Finally, systematic evaluation on tablet weight variation, content uniformity, friability, and dissolution using standard methods confirms the commercial manufacturability of FC tablets. Through this work, we have demonstrated the potential of integrated crystal and particle engineering in expediting the development of tablet products of challenging drugs using the economical direct compression process.

Design, synthesis, and characterization of new 5-fluorocytosine salts.

5-Fluorocytosine (FC), an antifungal drug and a cytosine derivative, has a complex solid-state landscape that challenges its development into a drug product. A total of eight new FC salts, both cytosinium and hemicytosinium, with four strong acids were prepared by controlling acid concentration in the crystallization medium. The pharmaceutically acceptable saccharin salt of FC exhibits superior phase stability and, hence, has the potential to address the instability problem of FC associated with hydration.

Comparison of MICs of fluconazole and flucytosine when dissolved in dimethyl sulfoxide or water.

A total of 145 clinical strains of Candida species were tested by the Clinical and Laboratory Standards Institute M27-A3 methodology to determine if replacing water with dimethyl sulfoxide as the solvent for fluconazole and flucytosine impacted the in vitro potency. No significant differences in MIC values were observed with either antifungal between the two solvents against any Candida species, and the essential agreement for each agent between the two solvents was greater than 99%.

Synthesis of cyclopentanyl carbocyclic 5-fluorocytosine ((-)-5-fluorocarbodine) using a facially selective hydrogenation approach.

An efficient synthetic route to biologically relevant (-)-5-fluorocarbodine 6 was developed. Direct coupling of N(6)-protected 5-fluorouracil 15 with cyclopentenyl intermediate 13, followed by formation of a macrocycle between the base and the carbocyclic sugar moiety, via ring-closing metathesis, allowed for a facial selective hydrogenation of the sugar double bond to give, exclusively, the desired 4'-ß stereoisomer.

Effects of genetically engineered stem cells expressing cytosine deaminase and interferon-beta or carboxyl esterase on the growth of LNCaP rrostate cancer cells.

The risk of prostate cancer has been increasing in men by degrees. To develop a new prostate cancer therapy, we used a stem cell-derived gene directed prodrug enzyme system using human neural stem cells (hNSCs) that have a tumor-tropic effect. These hNSCs were transduced with the therapeutic genes for bacterial cytosine deaminase (CD), alone or in combination with the one encoding human interferon-beta (IFN-ß) or rabbit carboxyl esterase (CE) to generate HB1.F3.CD, HB1.F3.CD.IFN-ß, and HB1.F3.CE cells, respectively. CD enzyme can convert the prodrug 5-fluorocytosine (5-FC) into the activated form 5-fluorouracil (5-FU). In addition, CE enzyme can convert the prodrug CPT-11 into a toxic agent, SN-38. In our study, the human stem cells were found to migrate toward LNCaP human prostate cancer cells rather than primary cells. This phenomenon may be due to interactions between chemoattractant ligands and receptors, such as VEGF/VEGFR2 and SCF/c-Kit, expressed as cancer and stem cells, respectively. The HB1.F3.CE, HB.F3.CD, or HB1.F3.CD.IFN-ß cells significantly reduced the LNCaP cell viability in the presence of the prodrugs 5-FC or CPT-11. These results indicate that stem cells expressing therapeutic genes can be used to develop a new strategy for selectively treating human prostate cancer.

Cocrystals of 5-fluorocytosine. I. Coformers with fixed hydrogen-bonding sites.

The antifungal drug 5-fluorocytosine (4-amino-5-fluoro-1,2-dihydropyrimidin-2-one) was cocrystallized with five complementary compounds in order to better understand its drug-receptor interaction. The first two compounds, 2-aminopyrimidine (2-amino-1,3-diazine) and N-acetylcreatinine (N-acetyl-2-amino-1-methyl-5H-imidazol-4-one), exhibit donor-acceptor sites for R(2)(2)(8) heterodimer formation with 5-fluorocytosine. Such a heterodimer is observed in the cocrystal with 2-aminopyrimidine (I); in contrast, 5-fluorocytosine and N-acetylcreatinine [which forms homodimers in its crystal structure (II)] are connected only by a single hydrogen bond in (III). The other three compounds 6-aminouracil (6-amino-2,4-pyrimidinediol), 6-aminoisocytosine (2,6-diamino-3H-pyrimidin-4-one) and acyclovir [acycloguanosine or 2-amino-9-[(2-hydroxyethoxy)methyl]-1,9-dihydro-6H-purin-6-one] possess donor-donor-acceptor sites; therefore, they can interact with 5-fluorocytosine to form a heterodimer linked by three hydrogen bonds. In the cocrystals with 6-aminoisocytosine (Va)-(Vd), as well as in the cocrystal with the antiviral drug acyclovir (VII), the desired heterodimers are observed. However, they are not formed in the cocrystal with 6-aminouracil (IV), where the components are connected by two hydrogen bonds. In addition, a solvent-free structure of acyclovir (VI) was obtained. A comparison of the calculated energies released during dimer formation helped to rationalize the preference for hydrogen-bonding interactions in the various cocrystal structures.

Cocrystals of 5-fluorocytosine. II. Coformers with variable hydrogen-bonding sites.

Two flexible molecules, biuret and 6-acetamidouracil, were cocrystallized with 5-fluorocytosine to study their conformational preferences. In the cocrystal with 5-fluorocytosine (I), biuret exhibits the same conformation as in its hydrate. In contrast, 6-acetamidouracil can adopt two main conformations depending on its crystal environment: in crystal (II) the trans form characterized by an intramolecular hydrogen bond is observed, while in the cocrystal with 5-fluorocytosine (III), the complementary binding induces the cis form. Three cocrystals of 6-methylisocytosine demonstrate that complementary binding enables the crystallization of a specific tautomer. In the cocrystals with 5-fluorocytosine, (IVa) and (IVb), only the 3H tautomer of 6-methylisocytosine is present, whereas in the cocrystal with 6-aminoisocytosine, (V), the 1H tautomeric form is adopted. The complexes observed in the cocrystals are stabilized by three hydrogen bonds similar to those constituting the Watson-Crick C·G base pair.