Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1).

NAD(P)H quinone oxidoreductase-1 (NQO1) is a homodimeric protein that acts as a detoxifying enzyme or as a chaperone protein. Dicourmarol interacts with NQO1 at the NAD(P)H binding site and can both inhibit enzyme activity and modulate the interaction of NQO1 with other proteins. We show that the binding of dicoumarol and related compounds to NQO1 generates negative cooperativity between the monomers. This does not occur in the presence of the reducing cofactor, NAD(P)H, alone. Alteration of Gly150 (but not Gly149 or Gly174) abolished the dicoumarol-induced negative cooperativity. Analysis of the dynamics of NQO1 with the Gaussian network model indicates a high degree of collective motion by monomers and domains within NQO1. Ligand binding is predicted to alter NQO1 dynamics both proximal to the ligand binding site and remotely, close to the second binding site. Thus, drug-induced modulation of protein motion might contribute to the biological effects of putative inhibitors of NQO1.

SAT2 Foot-and-Mouth Disease Virus Structurally Modified for Increased Thermostability.

Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for the potential use of such mutants in improving the stability of SAT2 vaccines in countries where FMD is endemic, which rely heavily on the maintenance of the cold chain, with potential improvement to the duration of immune responses.

Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases.

Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.

Efficacy of SAT2 Foot-and-Mouth Disease Vaccines Formulated with Montanide ISA 206B and Quil-A Saponin Adjuvants.

The effective control of foot-and-mouth disease (FMD) relies strongly on the separation of susceptible and infected livestock or susceptible livestock and persistently infected wildlife, vaccination, and veterinary sanitary measures. Vaccines affording protection against multiple serotypes for longer than six months and that are less reliant on the cold chain during handling are urgently needed for the effective control of FMD in endemic regions. Although much effort has been devoted to improving the immune responses elicited through the use of modern adjuvants, their efficacy is dependent on the formulation recipe, target species and administration route. Here we compared and evaluated the efficacy of two adjuvant formulations in combination with a structurally stabilized SAT2 vaccine antigen, designed to have improved thermostability, antigen shelf-life and longevity of antibody response. Protection mediated by the Montanide ISA 206B-adjuvanted or Quil-A Saponin-adjuvanted SAT2 vaccines were comparable. The Montanide ISA 206B-adjuvanted vaccine elicited a higher SAT2 neutralizing antibody response and three times higher levels of systemic IFN-γ responses at 14- and 28-days post-vaccination (dpv) were observed compared to the Quil-A Saponin-adjuvanted vaccine group. Interestingly, serum antibodies from the immunized animals reacted similarly to the parental vaccine virus and viruses containing mutations in the VP2 protein that simulate antigenic drift in nature.

Inherent biophysical stability of foot-and-mouth disease SAT1, SAT2 and SAT3 viruses.

Foot-and-mouth disease (FMD) virus (FMDV) isolates show variation in their ability to withstand an increase in temperature. The FMDV is surprisingly thermolabile, even though this virus is probably subjected to a strong extracellular selective pressure by heat in hot climate regions where FMD is prevalent. The three SAT serotypes, with their particularly low biophysical stability also only yield vaccines of low protective capacity, even with multiple booster vaccinations. The aim of the study was to determine the inherent biophysical stability of field SAT isolates. To characterise the biophysical stability of 20 SAT viruses from Southern Africa, the thermofluor assay was used to monitor capsid dissociation by the release of the RNA genome under a range of temperature, pH and ionic conditions. The SAT2 and SAT3 viruses had a similar range of thermostability of 48-54 °C. However, the SAT1 viruses had a wider range of thermostability with an 8 °C difference but with many viruses being unstable at 43-46 °C. The thermostable A-serotype A24 control virus had the highest thermostability of 55 °C with some SAT2 and SAT3 viruses of similar thermostability. There was a 10 °C difference between the most unstable SAT virus (SAT1/TAN/2/99) and the highly stable A24 control virus. SAT1 viruses were generally more stable compared to SAT2 and SAT3 viruses at the pH range of 6.7-9.1. The effect of ionic buffers on capsid stability showed that SAT1 and SAT2 viruses had an increased stability of 2-9 °C and 2-6 °C, respectively, with the addition of 1 M NaCl. This is in contrast to the SAT3 viruses, which did not show improved stabilisation after addition of 1 M or 0.5 M NaCl buffers. Some buffers showed differing results dependent on the virus tested, highlighting the need to test SAT viruses with different solutions to establish the most stabilising option for storage of each virus. This study confirms for the first time that more stable SAT field viruses are present in the southern Africa region. This could facilitate the selection of the most stable circulating field strains, for adaptation to cultured BHK-21 cells or manipulation by reverse genetics and targeted mutation to produce improved vaccine master seed viruses.

Anticancer effects of phytocannabinoids used with chemotherapy in leukaemia cells can be improved by altering the sequence of their administration.

Phytocannabinoids possess anticancer activity when used alone, and a number have also been shown to combine favourably with each other in vitro in leukaemia cells to generate improved activity. We have investigated the effect of pairing cannabinoids and assessed their anticancer activity in cell line models. Those most effective were then used with the common anti-leukaemia drugs cytarabine and vincristine, and the effects of this combination therapy on cell death studied in vitro. Results show a number of cannabinoids could be paired together to generate an effect superior to that achieved if the components were used individually. For example, in HL60 cells, the IC50 values at 48 h for cannabidiol (CBD) and tetrahydrocannabinol (THC) when used alone were 8 and 13 µM, respectively; however, if used together, it was 4 µM. Median-effect analysis confirmed the benefit of using cannabinoids in pairs, with calculated combination indices being <1 in a number of cases. The most efficacious cannabinoid-pairs subsequently synergised further when combined with the chemotherapy agents, and were also able to sensitise leukaemia cells to their cytotoxic effects. The sequence of administration of these drugs was important though; using cannabinoids after chemotherapy resulted in greater induction of apoptosis, whilst this was the opposite when the schedule of administration was reversed. Our results suggest that when certain cannabinoids are paired together, the resulting product can be combined synergistically with common anti-leukaemia drugs allowing the dose of the cytotoxic agents to be dramatically reduced yet still remain efficacious. Nevertheless, the sequence of drug administration is crucial to the success of these triple combinations and should be considered when planning such treatments.

Evaluation of immune responses of stabilised SAT2 antigens of foot-and-mouth disease in cattle.

Foot-and-mouth disease (FMD) vaccines with improved stability and less reliant on a cold-chain are needed to improve the longevity of immune responses elicited in animals. This is especially so for serotypes O and SAT2 which are unstable in mildly acidic pH conditions or at elevated temperatures leading to dissociation of the capsid (146S particle) and loss of immunogenicity. Previously, stabilised SAT2 viruses were generated by reverse genetic approaches and assessed in vitro and in vivo with a guinea pig trial. Here we investigated the efficacy and comparative immunological responses of two thermostable and wild-type SAT2 vaccines over 5months followed by challenge. We assessed humoral immune responses elicited in cattle in terms of total and neutralizing antibodies and IgG1/2 isotyping; and cell-mediated responses of IFN-γ as in vitro markers of protection. Whilst there were significant differences in total and neutralizing antibodies for the vSAT2-93H group compared to other vaccinated groups after the first vaccination, there were no significant differences after the second immunization. Following intra-dermolingual challenge all vaccinated groups were fully protected as determined by the absence of generalized lesions. These results provide proof that two vaccine doses, consisting of SAT2 antigen combined with ISA206B adjuvant, administered 4-6 weeks apart were able to protect animals up to 5months pv. Additionally, vSAT2-93Y had significantly higher levels of IFN-γ after challenge and had a lower clinical score indicative of better protection compared to other vaccinated groups and the importance of cell mediated responses and antigen stability in protection.

Naltrexone at low doses upregulates a unique gene expression not seen with normal doses: Implications for its use in cancer therapy.

It has been reported that lower doses of the opioid antagonist naltrexone are able to reduce tumour growth by interfering with cell signalling as well as by modifying the immune system. We have evaluated the gene expression profile of a cancer cell line after treatment with low-dose naltrexone (LDN), and assessed the effect that adapting treatment schedules with LDN may have on enhancing efficacy. LDN had a selective impact on genes involved with cell cycle regulation and immune modulation. Similarly, the pro-apoptotic genes BAD and BIK1 were increased only after LDN. Continuous treatment with LDN had little effect on growth in different cell lines; however, altering the treatment schedule to include a phase of culture in the absence of drug following an initial round of LDN treatment, resulted in enhanced cell killing. Furthermore, cells pre-treated with LDN were more sensitive to the cytotoxic effects of a number of common chemotherapy agents. For example, priming HCT116 with LDN before treatment with oxaliplatin significantly increased cell killing to 49±7.0 vs. 14±2.4% in cultures where priming was not used. Interestingly, priming with NTX before oxaliplatin resulted in just 32±1.8% cell killing. Our data support further the idea that LDN possesses anticancer activity, which can be improved by modifying the treatment schedule.

Inhibiting Heat Shock Proteins Can Potentiate the Cytotoxic Effect of Cannabidiol in Human Glioma Cells.

Cannabinoids possess a number of characteristics that make them putative anticancer drugs, and their value as such is currently being explored in a number of clinical studies. To further understand the roles that cannabinoids may have, we performed gene expression profiling in glioma cell lines cultured with cannabidiol (CBD) and/or Δ9-tetrahydrocannabinol (THC), and pursued targets identified by this screening. Results showed that a large number of genes belonging to the heat shock protein (HSP) super-family were up-regulated following treatment, specifically with CBD. Increases were observed both at the gene and protein levels and arose as a consequence of increased generation of ROS by CBD, and correlated with an increase in a number of HSP client proteins. Furthermore, increases impeded the cytotoxic effect of CBD; an effect that was improved by co-culture with pharmacalogical inhibitors of HSPs. Similarly, culturing glioma cells with CBD and HSP inhibitors increased radiosensitivity when compared to CBD-alone. Taken together, these data indicate that the cytotoxic effects of CBD can be diminished by HSPs that indirectly rise as a result of CBD use, and that the inclusion of HSP inhibitors in CBD treatment regimens can enhance the overall effect.

The combination of cannabidiol and Δ9-tetrahydrocannabinol enhances the anticancer effects of radiation in an orthotopic murine glioma model.

High-grade glioma is one of the most aggressive cancers in adult humans and long-term survival rates are very low as standard treatments for glioma remain largely unsuccessful. Cannabinoids have been shown to specifically inhibit glioma growth as well as neutralize oncogenic processes such as angiogenesis. In an attempt to improve treatment outcome, we have investigated the effect of Δ(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD) both alone and in combination with radiotherapy in a number of glioma cell lines (T98G, U87MG, and GL261). Cannabinoids were used in two forms, pure (P) and as a botanical drug substance (BDS). Results demonstrated a duration- and dose-dependent reduction in cell viability with each cannabinoid and suggested that THC-BDS was more efficacious than THC-P, whereas, conversely, CBD-P was more efficacious than CBD-BDS. Median effect analysis revealed all combinations to be hyperadditive [T98G 48-hour combination index (CI) at FU50, 0.77-1.09]. Similarly, pretreating cells with THC-P and CBD-P together for 4 hours before irradiation increased their radiosensitivity when compared with pretreating with either of the cannabinoids individually. The increase in radiosensitivity was associated with an increase in markers of autophagy and apoptosis. These in vitro results were recapitulated in an orthotopic murine model for glioma, which showed dramatic reductions in tumor volumes when both cannabinoids were used with irradiation (day 21: 5.5 ± 2.2 mm(3) vs. 48.7 ± 24.9 mm(3) in the control group; P < 0.01). Taken together, our data highlight the possibility that these cannabinoids can prime glioma cells to respond better to ionizing radiation, and suggest a potential clinical benefit for glioma patients by using these two treatment modalities.

Challenges and prospects for the control of foot-and-mouth disease: an African perspective.

The epidemiology of foot-and-mouth disease (FMD) in Africa is unique in the sense that six of the seven serotypes of FMD viruses (Southern African Territories [SAT] 1, SAT2, SAT3, A, O, and C), with the exception of Asia-1, have occurred in the last decade. Due to underreporting of FMD, the current strains circulating throughout sub-Saharan Africa are in many cases unknown. For SAT1, SAT2, and serotype A viruses, the genetic diversity is reflected in antigenic variation, and indications are that vaccine strains may be needed for each topotype. This has serious implications for control using vaccines and for choice of strains to include in regional antigen banks. The epidemiology is further complicated by the fact that SAT1, SAT2, and SAT3 viruses are maintained and spread by wildlife, persistently infecting African buffalo in particular. Although the precise mechanism of transmission of FMD from buffalo to cattle is not well understood, it is facilitated by direct contact between these two species. Once cattle are infected they may maintain SAT infections without the further involvement of buffalo. No single strategy for control of FMD in Africa is applicable. Decision on the most effective regional control strategy should focus on an ecosystem approach, identification of primary endemic areas, animal husbandry practices, climate, and animal movement. Within each ecosystem, human behavior could be integrated in disease control planning. Different regions in sub-Saharan Africa are at different developmental stages and are thus facing unique challenges and priorities in terms of veterinary disease control. Many science-based options targeting improved vaccinology, diagnostics, and other control measures have been described. This review therefore aims to emphasize, on one hand, the progress that has been achieved in the development of new technologies, including research towards improved tailored vaccines, appropriate vaccine strain selection, vaccine potency, and diagnostics, and how it relates to the conditions in Africa. On the other hand, we focus on the unique epidemiological, ecological, livestock farming and marketing, socioeconomic, and governance issues that constrain effective FMD control. Any such new technologies should have the availability of safe livestock products for trade as the ultimate goal.

In silico screening reveals structurally diverse, nanomolar inhibitors of NQO2 that are functionally active in cells and can modulate NF-κB signaling.

The National Cancer Institute chemical database has been screened using in silico docking to identify novel nanomolar inhibitors of NRH:quinone oxidoreductase 2 (NQO2). The inhibitors identified from the screen exhibit a diverse range of scaffolds and the structure of one of the inhibitors, NSC13000 cocrystalized with NQO2, has been solved. This has been used to aid the generation of a structure-activity relationship between the computationally derived binding affinity and experimentally measured enzyme inhibitory potency. Many of the compounds are functionally active as inhibitors of NQO2 in cells at nontoxic concentrations. To show this, advantage was taken of the NQO2-mediated toxicity of the chemotherapeutic drug CB1954. The toxicity of this drug is substantially reduced when the function of NQO2 is inhibited, and many of the compounds achieve this in cells at nanomolar concentrations. The NQO2 inhibitors also attenuated TNFα-mediated, NF-кB-driven transcriptional activity. The link between NQO2 and the regulation of NF-кB was confirmed by using short interfering RNA to NQO2 and by the observation that NRH, the cofactor for NQO2 enzyme activity, could regulate NF-кB activity in an NQO2-dependent manner. NF-кB is a potential therapeutic target and this study reveals an underlying mechanism that may be usable for developing new anticancer drugs.

Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1).

The synthesis is reported here of two novel series of inhibitors of human NAD(P)H quinone oxidoreductase-1 (NQO1), an enzyme overexpressed in several types of tumor cell. The first series comprises substituted symmetric dicoumarol analogues; the second series contains hybrid compounds where one 4-hydroxycoumarin system is replaced by a different aromatic moiety. Several compounds show equivalent or improved NQO1 inhibition over dicoumarol, both in the presence and in the absence of added protein. Further, correlation is demonstrated between the ability of these agents to inhibit NQO1 and computed binding affinity. We have solved the crystal structure of NQO1 complexed to a hybrid compound and find good agreement with the in silico model. For both MIA PaCa-2 pancreatic tumor cells and HCT116 colon cancer cells, dicoumarol shows the greatest toxicity of all compounds. Thus, we provide a computational, synthetic, and biological platform to generate competitive NQO1 inhibitors with superior pharmacological properties to dicoumarol. This will allow a more definitive study of NQO1 activity in cells, in particular, its drug activating/detoxifying properties and ability to modulate oncoprotein stability.

Enhancing the in vitro cytotoxic activity of Delta9-tetrahydrocannabinol in leukemic cells through a combinatorial approach.

Delta(9)-Tetrahydrocannabinol (THC) is the active metabolite of cannabis, which has demonstrable cytotoxic activity in vitro. In support of our previously published data, we have investigated the interactions between THC and anti-leukemia therapies and studied the role of the signalling pathways in mediating these effects. Results showed clear synergistic interactions between THC and the cytotoxic agents in leukemic cells. Additionally, exposure of cells to sub lethal levels of THC (1 microM) sensitised cells to these cytotoxic agents, by reducing IC(50) values by approximately 50%. Sensitisation appeared to be dependent upon the ability of THC to down regulate phosphorylated ERK, as cells dominantly expressive of MEK were not sensitised to the cytotoxic drugs by equi-molar amounts of THC. Overall, these results demonstrate for the first time that a combination approach with THC and established cytotoxic agents may enhance cell death in vitro. Additionally the MAPK/ERK pathway appears responsible in part for these effects.

The in vitro effects of CRE-decoy oligonucleotides in combination with conventional chemotherapy in colorectal cancer cell lines.

The cAMP response element consensus sequence directs the transcription of a wide range of genes. A 24-mer single-stranded cAMP response element decoy oligonucleotide (CDO) has been shown to compete with these sequences for binding transcription factors and therefore interferes with cAMP-induced gene transcription. We have examined the effect of this CDO alone and in combination with a range of common chemotherapeutic agents in colorectal cancer cell lines. CDO had a potent anti-proliferative effect in colorectal cell lines, yet, a similar enhancement of cell death was not observed. Simple drug-drug interaction studies showed that combining CDO with chemotherapy resulted in an enhancement of the antiproliferative effects. Furthermore, this cytostatic effect was protracted and associated with an increase in senescence-associated beta-galactosidase activity at pH 6. There is a possible role for p21(waf1) in mediating this effect, as the enhancement of cell growth inhibition was not observed in cells lacking the ability to correctly upregulate this protein. Additionally, significant decreases in cyclin-dependent kinase (CDK) 1 and CDK 4 function were seen in the responsive cells. These data provide a possible model of drug interaction in colorectal cell lines, which involves the complex interplay of the molecules regulating the cell cycle. Clinically, the cytostatic ability of CDO could improve and enhance the antiproliferative effects of conventional cytotoxic agents.