Manunya Nuth

Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation

Abstract Wambi, C., Sanzari, J., Wan, X. S., Nuth, M., Davis, J., Ko, Y. H., Sayers, C. M., Baran, M., Ware, J. H. and Kennedy, A. R. Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation. Radiat. Res. 169, 384–396 (2008). The purpose of this study was to determine whether a dietary supplement consisting of l-selenomethionine, vitamin C, vitamin E succinate, α-lipoic acid and N-acetyl cysteine could improve the survival of mice after total-body irradiation. Antioxidants significantly increased the 30-day survival of mice after exposure to a potentially lethal dose of X rays when given prior to or after animal irradiation. Pretreatment of animals with antioxidants resulted in significantly higher total white blood cell and neutrophil counts in peripheral blood at 4 and 24 h after 1 Gy and 8 Gy. Antioxidants were effective in preventing peripheral lymphopenia only after low-dose irradiation. Antioxidant supplementation was also associated with increased bone marrow cell counts after irradiation. Supplementation with antioxidants was associated with increased Bcl2 and decreased Bax, caspase 9 and TGF-β1 mRNA expression in the bone marrow after irradiation. Maintenance of the antioxidant diet was associated with improved recovery of the bone marrow after sublethal or potentially lethal irradiation. Taken together, oral supplementation with antioxidants appears to be an effective approach for radioprotection of hematopoietic cells and improvement of animal survival, and modulation of apoptosis is implicated as a mechanism for the radioprotection of the hematopoietic system by antioxidants.

Protective effects of dietary antioxidants on proton total-body irradiation-mediated hematopoietic cell and animal survival.

Abstract Dietary antioxidants have radioprotective effects after γ-radiation exposure that limit hematopoietic cell depletion and improve animal survival. The purpose of this study was to determine whether a dietary supplement consisting of l-selenomethionine, vitamin C, vitamin E succinate, α-lipoic acid and N-acetyl cysteine could improve survival of mice after proton total-body irradiation (TBI). Antioxidants significantly increased 30-day survival of mice only when given after irradiation at a dose less than the calculated LD50/30; for these data, the dose-modifying factor (DMF) was 1.6. Pretreatment of animals with antioxidants resulted in significantly higher serum total white blood cell, polymorphonuclear cell and lymphocyte cell counts at 4 h after 1 Gy but not 7.2 Gy proton TBI. Antioxidants significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGFβ1 and increased bone marrow cell counts and spleen mass after TBI. Maintenance of the antioxidant diet resulted in improved recovery of peripheral leukocytes and platelets after sublethal and potentially lethal TBI. Taken together, oral supplementation with antioxidants appears to be an effective approach for radioprotection of hematopoietic cells and improvement of animal survival after proton TBI.

Design of potent poxvirus inhibitors of the heterodimeric processivity factor required for viral replication.

Smallpox constitutes a major bioterrorism threat, which underscores the need to develop antiviral drugs for rapid response in the event of an attack. Viral processivity factors are attractive drug targets in being both specific and essential for their cognate DNA polymerases to synthesize extended strands of DNA. An in silico model of the vacinnia virus processivity factor, comprised of the A20 and D4 heterocomplex, was constructed and used for lead optimization of an indole-based scaffold identified earlier from a high-throughput screening. On the basis of this model, a new class of potent antivirals against vaccinia virus was designed and synthesized, of which two (24a and 24b) exhibited superior improvement over the parent scaffold (IC50 = 42 and 46 vs 82000 nM, respectively). The ability of 24a to suppress vaccinia DNA synthesis is supported by the inhibition of late viral gene expression, as well as by the diminished incorporation of bromodeoxyuridine into viral replication factories.

Effects of Proton Radiation Dose, Dose Rate and Dose Fractionation on Hematopoietic Cells in Mice

Abstract The present study evaluated the acute effects of radiation dose, dose rate and fractionation as well as the energy of protons in hematopoietic cells of irradiated mice. The mice were irradiated with a single dose of 51.24 MeV protons at a dose of 2 Gy and a dose rate of 0.05–0.07 Gy/min or 1 GeV protons at doses of 0.1, 0.2, 0.5, 1, 1.5 and 2 Gy delivered in a single dose at dose rates of 0.05 or 0.5 Gy/min or in five daily dose fractions at a dose rate of 0.05 Gy/min. Sham-irradiated animals were used as controls. The results demonstrate a dose-dependent loss of white blood cells (WBCs) and lymphocytes by up to 61% and 72%, respectively, in mice irradiated with protons at doses up to 2 Gy. The results also demonstrate that the dose rate, fractionation pattern and energy of the proton radiation did not have significant effects on WBC and lymphocyte counts in the irradiated animals. These results suggest that the acute effects of proton radiation on WBC and lymphocyte counts are determined mainly by the radiation dose, with very little contribution from the dose rate (over the range of dose rates evaluated), fractionation and energy of the protons.

Identification of Inhibitors that Block Vaccinia Virus Infection by Targeting the DNA Synthesis Processivity Factor D4

Smallpox was globally eradicated 30 years ago by vaccination. The recent threat of bioterrorism demands the development of improved vaccines and novel therapeutics to effectively preclude a reemergence of smallpox. One new therapeutic target is the vaccinia poxvirus processivity complex, comprising D4 and A20 proteins that enable the viral E9 DNA polymerase to synthesize extended strands. Five compounds identified from an AlphaScreen assay designed to disrupt A20:D4 binding were shown to be effective in: (i) blocking vaccinia processive DNA synthesis in vitro, (ii) preventing cellular infection with minimal cytotoxicity, and (iii) binding to D4, as evidenced by ThermoFluor. The EC(50) values for inhibition of viral infectivity ranged from 9.6 to 23 μM with corresponding selectivity indices (cytotoxicity CC(50)/viral infectivity EC(50)) of 3.9 to 17.8. The five compounds are thus potential therapeutics capable of halting smallpox DNA synthesis and infectivity through disruptive action against a component of the vaccinia processivity complex.

Induction of Cytokine Gene Expression in Human Thyroid Epithelial Cells Irradiated with HZE Particles (Iron Ions)

Abstract Gene expression profiles were examined using cDNA microarray technology in human thyroid epithelial (Htori-3) cells exposed to a low, non-toxic dose (10 cGy) of radiation from HZE particles in the form of iron ions in the absence or presence of selenomethionine (SeM). A total of 215 genes were differentially regulated 2 h after exposure to a 10-cGy dose of iron-ion radiation. In the microarray analysis, SeM had profound effects on the radiation-induced expression of several specific genes, which includes PLAU, IGFBP3, FOLR1, B4GALT1 and COL1A1. Of particular interest to us was a gene cluster, “secreted proteins”, that was up-regulated after radiation exposure. Seven up-regulated genes of this gene cluster fall within the chemokine/cytokine gene cluster, namely, CXCL1, CXCL2, IL6, IL11, IL8, IL24 and TGFβ2. In microarray studies, the radiation-induced up-regulated expression of some these genes encoding cytokine/chemokine proteins was significantly decreased by SeM treatment. For IL8, TGFβ2, CXCL1 and CXCL2, these observations were validated by qPCR techniques. It is concluded that SeM can regulate ionizing radiation-induced gene expression and may serve as an effective countermeasure for some of the acute inflammatory/immune responses induced by low-dose HZE-particle radiation.

Identification of Protein-Protein Interaction Inhibitors Targeting Vaccinia Virus Processivity Factor for Development of Antiviral Agents

ABSTRACT Poxvirus uracil DNA glycosylase D4 in association with A20 and the catalytic subunit of DNA polymerase forms the processive polymerase complex. The binding of D4 and A20 is essential for processive polymerase activity. Using an AlphaScreen assay, we identified compounds that inhibit protein-protein interactions between D4 and A20. Effective interaction inhibitors exhibited both antiviral activity and binding to D4. These results suggest that novel antiviral agents that target the protein-protein interactions between D4 and A20 can be developed for the treatment of infections with poxviruses, including smallpox.

Crystallization and preliminary X-ray diffraction analysis of three recombinant mutants of Vaccinia virus uracil DNA glycosylase.

Amino-acid residues located at a highly flexible area in the uracil DNA glycosylase of Vaccinia virus were mutated. In the crystal structure of wild-type D4 these residues lie at the dimer interface. Specifically, three mutants were generated: (i) residue Arg167 was replaced with an alanine (R167AD4), (ii) residues Glu171, Ser172 and Pro173 were substituted with three glycine residues (3GD4) and (iii) residues Glu171 and Ser172 were deleted (Δ171-172D4). Mutant proteins were expressed, purified and crystallized in order to investigate the effects of these mutations on the structure of the protein.

The processivity factor complex of feline herpes virus-1 is a new drug target.

Feline herpes virus-1 (FHV-1) is ubiquitous in the cat population and is a major cause of blindness for which antiviral drugs, including acyclovir, are not completely effective. Recurrent infections, due to reactivation of latent FHV-1 residing in the trigeminal ganglia, can lead to epithelial keratitis and stromal keratitis and eventually loss of sight. This has prompted the medical need for an antiviral drug that will specifically inhibit FHV-1 infection. A new antiviral target is the DNA polymerase and its associated processivity factor, which forms a complex that is essential for extended DNA strand synthesis. In this study we have cloned and expressed the FHV-1 DNA polymerase (f-UL30) and processivity factor (f-UL42) and demonstrated that both proteins are required to completely synthesize the 7249 nucleotide full-length DNA from the M13 primed-DNA template in vitro. Significantly, a known inhibitor of human herpes simplex virus-1 (HSV-1) processivity complex was shown to inhibit FHV-1 processive DNA synthesis in vitro and block infection of cells. This validates using f-UL42/f-UL30 as a new antiviral drug target to treat feline ocular herpes infection.

A Novel Target and Approach for Identifying Antivirals against Molluscum Contagiosum Virus

ABSTRACT The dermatological disease molluscum contagiosum (MC) presents as lesions restricted solely to the skin. The poxvirus molluscum contagiosum virus (MCV) is responsible for this skin disease that is easily transmitted through casual contact among all populations, with greater frequency in children and immunosuppressed individuals. In addition, sexual transmission of MCV in adolescents and adults is a health concern. Although the skin lesions ultimately resolve in immunocompetent individuals, they can persist for extended periods, be painful, and result in scarring. Treatment is problematic, and there is no drug that specifically targets MCV. The inability of MCV to propagate in cell culture has impeded drug development. To overcome these barriers, we integrated three new developments. First, we identified a new MCV drug target (mD4) that is essential for processive DNA synthesis in vitro. Second, we discovered a small chemical compound that binds to mD4 and prevents DNA synthesis in vitro. Third, and most significant, we engineered a hybrid vaccinia virus (mD4-VV) in which the natural vaccinia D4 (vD4) gene is replaced by the mD4 target gene. This hybrid virus is dependent on mD4 for viral growth in culture and is inhibited by the small compound. This target system provides, for the first time, a platform and approach for the discovery and evaluation of new therapeutics that can be used to treat MC.