Combined Effect of Basic Antiherpetic Drugs with a New Inhibitor of the Terminase Complex of Herpes Simplex Virus Type 1 in Vero Cell Cultures.

Carriers of herpes simplex virus type 1 (HSV-1) account for more than 90% of the global population. Infection manifests itself in the formation of blisters and ulcers on the face or genitals and can cause blindness, encephalitis, and generalized infection. All first- and second-line modern antiherpetic drugs selectively inhibit viral DNA polymerase. The purine-benzoxazine conjugate LAS-131 ((S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine), which we have described earlier, uses the large subunit of the HSV-1 terminase complex as a biotarget and selectively inhibits HSV-1 reproduction in vitro. Basically new results were for the first time obtained to characterize the combined effect on human herpesvirus infection for LAS-131 used in combination with practically significant antiviral compounds, including the nucleoside analogs acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), brivudine (BVdU), iododeoxyuridine (IdU), and adenine arabinoside (Ara-A); the nucleoside phosphonate analog cidofovir (CDV); and the pyrophosphate analog foscarnet (FOS). A cytopathic effect (CPE) inhibition assay showed that the drug concentration that inhibited the virus-induced CPE by 50% decreased by a factor of 2 (an additive effect, FOS) or more (a synergistic effect; ACV, PCV, GCV, IdU, BVdU, Ara-A, and CDV) when the drugs were used in combination with LAS-131. Nonpermissive conditions for HSV-1 reproduction were thus created at lower drug concentrations, opening up new real possibilities to control human herpesvirus infection.

Comparison of Famciclovir, Valaciclovir, and Brivudine Treatments in Adult Immunocompetent Patients With Herpes Zoster.

Herpes zoster (HZ) is a common disease characterized by the recurrence of varicella zoster, that stays dormant in sensory ganglia. The primary goal of this study was to compare efficiencies of famciclovir, valaciclovir, and brivudine in terms of pain relief in HZ patients. Records of patients who were admitted to the Dermatology Clinic of our hospital due to acute HZ between the years 2012 and 2014 were retrospectively analyzed. Treatment decisions were at the discretion of caring physicians as valaciclovir (VACV), famciclovir (FCV), and brivudine (BRV) based on the clinical observations. BRV, FCV, and VACV were effective in treating pain in acute HZ. There was no significant difference between mild and moderate HZ patients. In severe cases, a significant reduction in intensity of pain was observed on day 3 in the BRV group, on day 7 in the FCV group, and at 2-3 weeks in the VACV group. There were no significant side effects observed in any of the groups. Results of this study indicate that brivudine may be the first choice in severe HZ cases as it controls pain earlier and is easier to use because of its once daily administration.

ProTides of BVdU as potential anticancer agents upon efficient intracellular delivery of their activated metabolites.

Nucleosides represent a major chemotherapeutic class for treating cancer, however their limitations in terms of cellular uptake, nucleoside kinase-mediated activation and catabolism are well-documented. The monophosphate pro-nucleotides known as ProTides represents a powerful strategy for bypassing the dependence on active transport and nucleoside kinase-mediated activation. Herein, we report the structural tuning of BVdU ProTides. Forty six phosphoramidates were prepared and biologically evaluated against three different cancer cell lines; murine leukemia (L1210), human CD4+ T-lymphocyte (CEM) and human cervical carcinoma (HeLa). Twenty-fold potency enhancement compared to BVdU was achieved against L1210 cells. Interestingly, a number of ProTides showed low micromolar activity against CEM and HeLa cells compared to the inactive parent BVdU. The ProTides showed poor, if any measurable toxicity to non-tumourigenic human lung fibroblast cell cultures. Separation of four pairs of the diastereoisomeric mixtures and comparison of their spectral properties, biological activities and enzymatic activation rate is reported.

Spectrum of activity and mechanisms of resistance of various nucleoside derivatives against gammaherpesviruses.

The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-ß-D-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2'-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase.

Increased density of cutaneous nerve fibres in the affected dermatomes after herpes zoster therapy.

Herpes zoster neural injury was assessed by determining cutaneous nerve density in skin biopsies from the affected dermatomes of 35 adult patients with herpes zoster in the acute phase and 3 months post-treatment, using protein gene product 9.5 immunohistochemistry. In contrast to the significant increase in subepidermal nerve fibre density (11.77 ± 4.88/mm vs. 13.29 ± 5.74/mm, p = 0.045) after 3 months, no differences were found in epidermal free nerve endings (2.43 ± 2.35/mm and 2.8 ± 2.86/mm, p = 0.168). Patients with post-herpetic neuralgia had significantly lower subepidermal nerve fibre densities (9.7 ± 2.05/mm vs. 14.72 ± 6.13/mm, p = 0.011) compared with non-post-herpetic neuralgia patients. No differences in cutaneous nerve density were found in relation to antiviral therapy. In conclusion, 3 months after acute infection, no sign of epidermal innervation recovery is observed, while the increased subepidermal nerve fibre density in the affected dermatomes probably reflects nerve regeneration that is not affected by antiviral agent type. Subepidermal nerve fibre density is decreased in patients with post-herpetic neuralgia 3-months post-acute herpes zoster infection.

Efficacy and safety of brivudine for the treatment of herpes zoster: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVE: Brivudine has been used in herpes zoster (HZ) treatment for years, but the safety and efficacy of brivudine are inconclusive. Here we perform a meta-analysis to assess the efficacy, safety, incidence of postherpetic neuralgia of brivudine. METHODS: Data of randomized controlled Trials (RCTS) were obtained from the databases of both English (PubMed, Embase, and Cochrane Library) and Chinese (China National Knowledge Infrastructure, China Science Journal Database, and WanFang Database) literatures from inception to 12 September 2022. Meta-analyses of efficacy and safety of Brivudine for the treatment of herpes zoster for RCTS were conducted. RESULTS: The analyses included seven RCTS (2095 patients in experimental group and 2076 patients in control group) in the treatment of HZ with brivudine. It suggested that the brivudine group was superior to the control group in terms of efficacy (p = .0002) and incidence of postherpetic neuralgia (p = .04). But the incidence of adverse reactions has no significant difference between the brivudine and the control groups (p = .22). In addition, subgroup analysis of adverse events also showed that brivudine was about the same safety as other modalities in the treatment of HZ (p > .05). CONCLUSIONS: Brivudine is effective for HZ. However, the evidence on the safety of brivudine is insufficient.

Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: implications for the clinical efficacy of nucleoside analogues.

In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (Km=473 µM) and deoxyuridine (Km=578 µM), it prefers uridine (Km=92 µM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NP-I class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.

Phenotypic and genetic characterization of varicella-zoster virus mutants resistant to acyclovir, brivudine and/or foscarnet.

The treatment of varicella-zoster virus (VZV) reactivation is based on nucleoside analogues acyclovir (ACV) and bromevinyldeoxyuridine (BVdU) and a phosphonic acid derivative (PFA). Drug-resistant mutants of 3 wild-type (WT) VZV strains were obtained by exposure of human retinal pigment epithelial (hRPE) cells inoculated with cell-free WT virus at increasing concentrations of ACV, BVdU, and PFA. In addition to single-drug resistance, a cross-resistance of isolates vs. ACV was observed for PFA-resistant strains. Single-nucleotide (nt) exchanges resulting in amino acid (aa) substitutions were observed within the DNA polymerase (ORF 28) and/or thymidine kinase (ORF 36) of 3 of 3 ACV-, 2 of 3 BVdU-, and 3 of 3 PFA-resistant strains. Interestingly, aa substitutions were also observed within the immediate-early regulatory protein and major transactivator IE 62 (ORF 62), and the envelope glycoprotein (g) I (ORF 67) of the BVdU-resistant mutant of strain PP. No aa substitutions were observed in the protein sequences of gene products encoded by ORF 5 (gK, a glycoprotein arranging exocytosis of viral-loaded vacuoles), ORF 14 (gC), ORF 31 (gB), ORF 37 (gH), ORF 47 (protein kinase, involved in major phosphorylating processes), ORF 60 (gL, important for syncytia forming of infected cells in combination with gH), ORF 63 (major transactivator, part of the tegument), and ORF 68 (gE, triggers fusion of viral loaded vacuoles with cell membranes by heterodimerizing with gI). Phenotypic analysis revealed a slow-growth phenotype and a formation of smaller plaques of resistant mutants. Future studies should prove the presence of those resistant mutants in herpes zoster patients and the potential consequences of their putative reduced fitness on the success of therapeutical interventions.

Crystal structure of poxvirus thymidylate kinase: an unexpected dimerization has implications for antiviral therapy.

Unlike most DNA viruses, poxviruses replicate in the cytoplasm of host cells. They encode enzymes needed for genome replication and transcription, including their own thymidine and thymidylate kinases. Some herpes viruses encode only 1 enzyme catalyzing both reactions, a peculiarity used for prodrug activation to obtain maximum specificity. We have solved the crystal structures of vaccinia virus thymidylate kinase bound to TDP or brivudin monophosphate. Although the viral and human enzymes have similar sequences (42% identity), they differ in their homodimeric association and active-site geometry. The vaccinia TMP kinase dimer arrangement is orthogonal and not antiparallel as in human enzyme. This different monomer orientation is related to the presence of a canal connecting the edge of the dimer interface to the TMP base binding pocket. Consequently, the pox enzyme accommodates nucleotides with bulkier bases, like brivudin monophosphate and dGMP; these are efficiently phosphorylated and stabilize the enzyme. The brivudin monophosphate-bound structure explains the structural basis for this specificity, opening the way to the rational development of specific antipox agents that may also be suitable for poxvirus TMP kinase gene-based chemotherapy of cancer.

New Mechanism of Acyclovir Resistance in Herpes Simplex Virus 1, Which Has a UAG Stop Codon between the First and Second AUG Initiation Codons.

Morphological changes in the structure of the herpes simplex virus 1 (HSV-1) viral thymidine kinase (vTK) polypeptide usually lead to conferring acyclovir (ACV) resistance. HSV-1 I4-2, in which a UAG stop codon is present at the 8th position between the 1st initiation AUG codon (1st position) and the 2nd initiation AUG codon (46th position) of the HSV-1 vTK gene, showed sensitivity to ACV. In contrast, HSV-1 KG111, in which a UAG stop codon was artificially inserted at the 44th position, showed resistance to ACV at 39˚C. The mechanism underlying the difference in the sensitivity profiles was elucidated. The virus recombinants HSV-1-TK(8UAG) and HSV-1-TK(44UAG) containing a UAG stop codon at the 8th and 44th positions counted from the 1st initiation codon, respectively, were generated and tested for susceptibility to antiviral compounds. HSV-1-TK(8UAG) and HSV-1-TK(44UAG) were sensitive and resistant to ACV and BVdU at 37˚C, respectively. The expression level of the truncated vTK translated from the 2nd initiation codon in Vero cells infected with HSV-1-TK(44UAG) was clearly less than that with HSV-1-TK(8UAG) in a temperature-dependent manner. The differences in the antiviral sensitivity profiles were due to the position of the UAG stop codon between the 1st and the 2nd initiation codons.

Acute hepatitis due to brivudin: a case report.

BACKGROUND/AIMS: Brivudin is licensed in several European countries for the treatment of herpetic infections, and is considered safe (approximately 1% of patients with transient elevation of liver enzymes) in large multicenter trials. METHODS: We report a case of acute brivudin hepatitis documented with a liver biopsy in detail. RESULTS: Liver biopsy demonstrated acute liver injury with a predominant cytolytic pattern and features suggestive of a drug-induced immunoallergic hepatitis. Elevated ALT levels returned to normal within weeks. CONCLUSIONS: This is the first published case of acute immunoallergic hepatitis due to brivudin.

High efficiency latency and activation of herpes simplex virus in human cells.

Herpes simplex virus (HSV) exists in humans in a latent form that can be activated. To characterize the molecular basis of the cell-virus interactions and to analyze the state of the latent HSV genome, an in vitro model system was established. In this system a large fraction of the latently infected cells contain an HSV genome that can be activated. Cell survival was reduced minimally after repression of high multiplicity HSV type 1 (HSV-1) infection of human fibroblast cells with (E)-5-(2-bromovinyl)-2'-deoxyuridine in combination with human leukocyte interferon (IFN-alpha). A minimum of 1 to 3 percent of the surviving cells contained an HSV genome that could be activated either by human cytomegalovirus superinfection or reduction in incubation temperature.

Epstein-Barr virus: inhibition of replication by three new drugs.

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with three human malignancies. Acyclovir [9-(2-hydroxyethoxymethyl)guanine], the first clinically useful drug effective against replication of EBV, is without effect against latent or persistent EBV infection. Three nucleoside analogs, E-5-(2-bromovinyl)-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine, and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil are potent inhibitors of EBV replication in vitro. Moreover, in contrast to the reversibility of viral inhibition by Acyclovir, these three drugs have prolonged effects in suppressing viral replication even after the drugs are removed from persistently infected cell cultures.

Inhibition of homologous recombination by treatment with BVDU (brivudin) or by RAD51 silencing increases chromosomal damage induced by bleomycin in mismatch repair-deficient tumour cells.

Mismatch repair (MMR) has been shown to control homologous recombination (HR) by aborting strand exchange between divergent sequences. We previously demonstrated that MMR-deficient tumour cells are more resistant to chromosomal damage induced by bleomycin (BLM) during the G(2) phase, likely due to the lack of the MMR inhibitory effect on HR. Aim of this study was to investigate whether inhibition of HR by the nucleoside analogue BVDU [(E)-5(2-bromovinyl)-2'-deoxyuridine, brivudin], or silencing of genes involved in HR function, might affect sensitivity of MMR-deficient tumour cells to DNA damage induced by BLM in G(2). The results indicated that BVDU increased chromatid damage and DNA double strand breaks induced by BLM only in MMR-deficient MT-1, HL-60R, HCT116 cells, which are more resistant to BLM with respect to MMR-proficient TK-6, HL-60S and HCT116/3-6 lines. Silencing of RAD51, a key component of HR, increased sensitivity of MMR-deficient HCT-15 cells to BLM clastogenicity; in this case combined treatment with BVDU had no additional effect. Similarly, treatment with BVDU did not affect BLM clastogenicity in CAPAN-1 cells, characterized by a defective HR due to BRCA2 mutations. Conversely, BVDU increased chromatid breaks induced by BLM in HCT-15 cells transiently silenced for DNA-PK catalytic subunit, which plays a key role in non-homologous end joining. The BVDU-mediated increase of chromatid breaks in MMR-deficient cells did not depend on its previously reported inhibitory effect on poly(ADP-ribose) polymerase (PARP). In fact, it was observed also in cells stably silenced for PARP-1, which is responsible for most of cellular PARP activity. These data support the suggestion that the higher sensitivity of MMR-proficient versus MMR-deficient cells to BLM-induced chromatid breaks in the G(2) phase is a consequence of the inhibition of HR by MMR. In MMR-deficient cells, BVDU attenuates the repair of BLM-induced DSBs and this is likely to occur via inhibition of HR.

Separating host and microbiome contributions to drug pharmacokinetics and toxicity.

The gut microbiota is implicated in the metabolism of many medical drugs, with consequences for interpersonal variation in drug efficacy and toxicity. However, quantifying microbial contributions to drug metabolism is challenging, particularly in cases where host and microbiome perform the same metabolic transformation. We combined gut commensal genetics with gnotobiotics to measure brivudine drug metabolism across tissues in mice that vary in a single microbiome-encoded enzyme. Informed by these measurements, we built a pharmacokinetic model that quantitatively predicts microbiome contributions to systemic drug and metabolite exposure, as a function of bioavailability, host and microbial drug-metabolizing activity, drug and metabolite absorption, and intestinal transit kinetics. Clonazepam studies illustrate how this approach disentangles microbiome contributions to metabolism of drugs subject to multiple metabolic routes and transformations.

A life-threatening drug-drug interaction between capecitabine and brivudine in a patient with metastatic breast cancer.

In the current report we present the case of a patient experiencing a life-threatening drug-drug interaction involving the concurrent administration of capecitabine and brivudine. A 65- year-old female with metastatic breast cancer was commenced on brivudine for Herpes Zoster, while on capecitabine treatment, by a physician unfamiliar with the potential repercussions of this drug-drug interaction. As a result, she developed skin rash, severe oral mucositis, and severe and prolonged pancytopenia. These side effects were attributed to a serious interaction of capecitabine with brivudine, resulting in inhibition of dihydropyrimidine dehydrogenase. The patient was admitted for supportive care including intravenous hydration, parenteral nutrition, mouth care solutions, fluconazole, antimicrobial therapy, filgrastim, red blood cell and platelet transfusions. She successfully recovered and was discharged on the 26th day after her admission. Drug-drug interactions can be serious, even life-threatening; thus the physicians should be cautious when prescribing new drugs.

Engineered Thymidine-Active Deoxycytidine Kinase for Bystander Killing of Malignant Cells.

Suicide transgenes encode proteins that are either capable of activating specific prodrugs into cytotoxic antimetabolites that can trigger cancer cell apoptosis or are capable of directly inducing apoptosis. Suicide gene therapy of cancer (SGTC) involves the targeted or localized delivery of suicide transgene sequences into tumor cells by means of various gene delivery vehicles. SGTC that operates via the potentiation of small-molecule pharmacologic agents can elicit the elimination of cancer cells within a tumor beyond only those cells successfully transduced. Such "bystander effects ", typically mediated by the spread of activated cytotoxic antimetabolites from the transduced cells expressing the suicide transgene to adjacent cells in the tumor, can lead to a significant reduction of the tumor mass without the requirement of transduction of a high percentage of cells within the tumor. The spread of activated cytotoxic molecules to adjacent cells is mediated primarily by diffusion and normally involves gap junctional intercellular communications (GJIC). We have developed a novel SGTC system based on viral vector-mediated delivery of an engineered variant of human deoxycytidine kinase (dCK), which is capable of phosphorylating uridine- and thymidine-based nucleoside analogues that are not substrates for wild-type dCK, such as bromovinyl deoxyuridine (BVdU) and L-deoxythymidine (LdT). Since our dCK-based SGTC system is capable of mediating strong bystander cell killing, it holds promise for clinical translation. In this chapter, we detail the key procedures for the preparation of recombinant lentivectors for the delivery of engineered dCK, transduction of tumor cells, and evaluation of bystander cell killing effects in vitro and in vivo.

Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (dNK; EC 2.7.1.145) has a high turnover rate and a wide substrate range that makes it a very good candidate for gene therapy. This concept is based on introducing a suicide gene into malignant cells in order to activate a prodrug that eventually may kill the cell. To be able to optimize the function of dNK, it is vital to have structural information of dNK complexes. In this study we present crystal structures of dNK complexed with four different nucleoside analogs (floxuridine, brivudine, zidovudine and zalcitabine) and relate them to the binding of substrate and feedback inhibitors. dCTP and dGTP bind with the base in the substrate site, similarly to the binding of the feedback inhibitor dTTP. All nucleoside analogs investigated bound in a manner similar to that of the pyrimidine substrates, with many interactions in common. In contrast, the base of dGTP adopted a syn-conformation to adapt to the available space of the active site.

Synthesis of nucleoside-based antiviral drugs in ionic liquids.

Nucleoside-based antiviral drugs have been synthesized using imidazolium-based ionic liquids as reaction medium. The ionic liquids were proved to be better solvents for all the nucleoside in terms of solubility and reaction medium as compared to conventional molecular solvents.