Triazoxins: Novel nucleosides with anti-Giardia activity.

Novel nucleoside analogues named "triazoxins" were synthesized. Of these, two analogues were found to be highly effective against Giardia lamblia, an intestinal parasite and a major cause of waterborne infection, worldwide. While compound 7 reduced the growth of trophozoites in culture (IC50, ~5 µM), compound 21 blocked the in vitro cyst production (IC50 ~5 µM). Compound 21 was also effective against trophozoites (IC50, ~36 µM). A third analogue (compound 8) was effective against both trophozoites (IC50, ~36 µM) and cysts (IC50, ~20 µM) although at higher concentration. Thus triazoxin analogues are unique and exhibit morphology (i.e., trohozoites or cysts) -specific effects against Giardia.

Dual anti-HIV mechanism of clofarabine.

BACKGROUND: HIV-1 replication kinetics inherently depends on the availability of cellular dNTPs for viral DNA synthesis. In activated CD4(+) T cells and other rapidly dividing cells, the concentrations of dNTPs are high and HIV-1 reverse transcription occurs in an efficient manner. In contrast, nondividing cells such as macrophages have lower dNTP pools, which restricts efficient reverse transcription. Clofarabine is an FDA approved ribonucleotide reductase inhibitor, which has shown potent antiretroviral activity in transformed cell lines. Here, we explore the potency, toxicity and mechanism of action of clofarabine in the human primary HIV-1 target cells: activated CD4(+) T cells and macrophages. RESULTS: Clofarabine is a potent HIV-1 inhibitor in both activated CD4(+) T cells and macrophages. Due to its minimal toxicity in macrophages, clofarabine displays a selectivity index over 300 in this nondividing cell type. The anti-HIV-1 activity of clofarabine correlated with a significant decrease in both cellular dNTP levels and viral DNA synthesis. Additionally, we observed that clofarabine triphosphate was directly incorporated into DNA by HIV-1 reverse transcriptase and blocked processive DNA synthesis, particularly at the low dNTP levels found in macrophages. CONCLUSIONS: Taken together, these data provide strong mechanistic evidence that clofarabine is a dual action inhibitor of HIV-1 replication that both limits dNTP substrates for viral DNA synthesis and directly inhibits the DNA polymerase activity of HIV-1 reverse transcriptase.

5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine.

5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.

Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase.

Although many compounds have been approved for the treatment of human immunodeficiency type-1 (HIV-1) infection, additional anti-HIV-1 drugs (particularly those belonging to new drug classes) are still needed due to issues such as long-term drug-associated toxicities, transmission of drug-resistant variants, and development of multi-class resistance. Lethal mutagenesis represents an antiviral strategy that has not yet been clinically translated for HIV-1 and is based on the use of small molecules to induce excessive levels of deleterious mutations within the viral genome. Here, we show that 5-azacytidine (5-aza-C), a ribonucleoside analog that induces the lethal mutagenesis of HIV-1, and multiple inhibitors of the enzyme ribonucleotide reductase (RNR) interact in a synergistic fashion to more effectively reduce the infectivity of HIV-1. In these drug combinations, RNR inhibitors failed to significantly inhibit the conversion of 5-aza-C to 5-aza-2'-deoxycytidine, suggesting that 5-aza-C acts primarily as a deoxyribonucleoside even in the presence of RNR inhibitors. The mechanism of antiviral synergy was further investigated for the combination of 5-aza-C and one specific RNR inhibitor, resveratrol, as this combination improved the selectivity index of 5-aza-C to the greatest extent. Antiviral synergy was found to be primarily due to the reduced accumulation of reverse transcription products rather than the enhancement of viral mutagenesis. To our knowledge, these observations represent the first demonstration of antiretroviral synergy between a ribonucleoside analog and RNR inhibitors, and encourage the development of additional ribonucleoside analogs and RNR inhibitors with improved antiretroviral activity.

Lack of mutational hot spots during decitabine-mediated HIV-1 mutagenesis.

Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity.

Discovery of novel ribonucleoside analogs with activity against human immunodeficiency virus type 1.

Reverse transcription is an important early step in retrovirus replication and is a key point targeted by evolutionarily conserved host restriction factors (e.g., APOBEC3G, SamHD1). Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a major target of antiretroviral drugs, and concerns regarding drug resistance and off-target effects have led to continued efforts for identifying novel approaches to targeting HIV-1 RT. Several observations, including those obtained from monocyte-derived macrophages, have argued that ribonucleotides and their analogs can, intriguingly, impact reverse transcription. For example, we have previously demonstrated that 5-azacytidine has its greatest antiviral potency during reverse transcription by enhancement of G-to-C transversion mutations. In the study described here, we investigated a panel of ribonucleoside analogs for their ability to affect HIV-1 replication during the reverse transcription process. We discovered five ribonucleosides-8-azaadenosine, formycin A, 3-deazauridine, 5-fluorocytidine, and 2'-C-methylcytidine-that possess anti-HIV-1 activity, and one of these (i.e., 3-deazauridine) has a primary antiviral mechanism that involves increased HIV-1 mutational loads, while quantitative PCR analysis determined that the others resulted in premature chain termination. Taken together, our findings provide the first demonstration of a series of ribonucleoside analogs that can target HIV-1 reverse transcription with primary antiretroviral mechanisms that include premature termination of viral DNA synthesis or enhanced viral mutagenesis.

Novel inhibitors of human immunodeficiency virus type 2 infectivity.

Human immunodeficiency virus type 2 (HIV-2) infects about two million people worldwide. HIV-2 has fewer treatment options than HIV-1, yet may evolve drug resistance more quickly. We have analysed several novel drugs for anti-HIV-2 activity. It was observed that 5-azacytidine, clofarabine, gemcitabine and resveratrol have potent anti-HIV-2 activity. The EC50 values for 5-azacytidine, clofarabine and resveratrol were found to be significantly lower with HIV-2 than with HIV-1. A time-of-addition assay was used to analyse the ability of these drugs to interfere with HIV-2 replication. Reverse transcription was the likely target for antiretroviral activity. Taken together, several novel drugs have been discovered to have activity against HIV-2. Based upon their known activities, these drugs may elicit enhanced HIV-2 mutagenesis and therefore be useful for inducing HIV-2 lethal mutagenesis. In addition, the data are consistent with HIV-2 reverse transcriptase being more sensitive than HIV-1 reverse transcriptase to dNTP pool alterations.

5,6-Dihydro-5-aza-2'-deoxycytidine potentiates the anti-HIV-1 activity of ribonucleotide reductase inhibitors.

The nucleoside analog 5,6-dihydro-5-aza-2'-deoxycytidine (KP-1212) has been investigated as a first-in-class lethal mutagen of human immunodeficiency virus type-1 (HIV-1). Since a prodrug monotherapy did not reduce viral loads in Phase II clinical trials, we tested if ribonucleotide reductase inhibitors (RNRIs) combined with KP-1212 would improve antiviral activity. KP-1212 potentiated the activity of gemcitabine and resveratrol and simultaneously increased the viral mutant frequency. G-to-C mutations predominated with the KP-1212-resveratrol combination. These observations represent the first demonstration of a mild anti-HIV-1 mutagen potentiating the antiretroviral activity of RNRIs and encourage the clinical translation of enhanced viral mutagenesis in treating HIV-1 infection.

Activity of a novel combined antiretroviral therapy of gemcitabine and decitabine in a mouse model for HIV-1.

The emergence of drug resistance threatens to limit the use of current anti-HIV-1 drugs and highlights the need to expand the number of treatment options available for HIV-1-infected individuals. Our previous studies demonstrated that two clinically approved drugs, decitabine and gemcitabine, potently inhibited HIV-1 replication in cell culture through a mechanism that is distinct from the mechanisms for the drugs currently used to treat HIV-1 infection. We further demonstrated that gemcitabine inhibited replication of a related retrovirus, murine leukemia virus (MuLV), in vivo using the MuLV-based LP-BM5/murine AIDS (MAIDS) mouse model at doses that were not toxic. Since decitabine and gemcitabine inhibited MuLV and HIV-1 replication with similar potency in cell culture, the current study examined the efficacy and toxicity of the drug combination using the MAIDS model. The data demonstrate that the drug combination inhibited disease progression, as detected by histopathology, viral loads, and spleen weights, at doses lower than those that would be required if the drugs were used individually. The combination of decitabine and gemcitabine exerted antiviral activity at doses that were not toxic. These findings indicate that the combination of decitabine and gemcitabine shows potent antiretroviral activity at nontoxic doses and should be further investigated for clinical relevance.

Discovery of drugs that possess activity against feline leukemia virus.

Feline leukemia virus (FeLV) is a gammaretrovirus that is a significant cause of neoplastic-related disorders affecting cats worldwide. Treatment options for FeLV are limited, associated with serious side effects, and can be cost-prohibitive. The development of drugs used to treat a related retrovirus, human immunodeficiency virus type 1 (HIV-1), has been rapid, leading to the approval of five drug classes. Although structural differences affect the susceptibility of gammaretroviruses to anti-HIV drugs, the similarities in mechanism of replication suggest that some anti-HIV-1 drugs may also inhibit FeLV. This study demonstrates the anti-FeLV activity of four drugs approved by the US FDA (Food and Drug Administration) at non-toxic concentrations. Of these, tenofovir and raltegravir are anti-HIV-1 drugs, while decitabine and gemcitabine are approved to treat myelodysplastic syndromes and pancreatic cancer, respectively, but also have anti-HIV-1 activity in cell culture. Our results indicate that these drugs may be useful for FeLV treatment and should be investigated for mechanism of action and suitability for veterinary use.

Anti-HIV-1 activity of resveratrol derivatives and synergistic inhibition of HIV-1 by the combination of resveratrol and decitabine.

Ribonucleotide reductase inhibitors enhance the anti-HIV-1 activities of a variety of nucleoside analogs, including those that act as chain terminators and those that increase the HIV-1 mutation rate. However the use of these ribonucleotide reductase inhibitors is limited by their associated toxicities. The hydroxylated phytostilbene resveratrol has activity in a host of systems including inhibition of ribonucleotide reductase and has minimal toxicity. Here we synthesized derivatives of resveratrol and examined them for anti-HIV-1 activity and their ability to enhance the antiviral activity of decitabine, a nucleoside analog that decreases viral replication by increasing the HIV-1 mutation rate. The data demonstrates that six of the derivatives have anti-HIV-1 activity greater than resveratrol. However, only resveratrol acted in synergy with decitabine to inhibit HIV-1 infectivity. These results reveal novel resveratrol derivatives with anti-HIV-1 activity that may have mechanisms of action that differ from the drugs currently used to treat HIV-1.

Cyanide toxicity in juvenile pigs and its reversal by a new prodrug, sulfanegen sodium.

BACKGROUND: Cyanide (CN) toxicity is a serious clinical problem and can occur with sodium nitroprusside (SNP) administration, accidental smoke inhalation, industrial mishaps, and bio-terrorism. In this study, we induced severe CN toxicity independently with SNP or sodium cyanide (NaCN) in a juvenile pig model to demonstrate reversal of severe CN toxicity with a new antidote, sulfanegen sodium, a prodrug of 3-mercaptopyruvate. METHODS: SNP study: A pilot study in 11 anesthetized, mechanically ventilated juvenile pigs allowed us to determine the dose of SNP to induce CN toxicity. Blood CN, serum lactates, and blood gases were monitored. CN toxicity was defined as the occurrence of severe lactic acidosis accompanied by significant elevation in blood CN levels. Based on this pilot study, 8 anesthetized pigs received a high-dose i.v. infusion of SNP (100 mg/h) for 2 hours to induce CN toxicity. They were then randomized to receive either sulfanegen sodium or placebo. Four pigs received 3 doses of sulfanegen sodium (2.5 g i.v.) every hour after induction of severe CN toxicity, and 4 pigs received placebo. NaCN study: A pilot study was conducted in 4 spontaneously ventilating pigs sedated with propofol plus ketamine to demonstrate hemodynamic and metabolic stability for several hours. After this, 6 pigs were similarly sedated and given NaCN in bolus aliquots to produce CN toxicity ultimately resulting in death. Hemodynamics and metabolic variables were followed to define peak CN toxicity. In another group of 6 pigs, severe CN toxicity was induced by this method, and at peak toxicity, the animals were given sulfanegen sodium (2.5 g i.v.) followed by a repeat dose 60 minutes later in surviving animals. RESULTS: SNP study: The pilot study demonstrated the occurrence of a significant increase in blood CN levels (P < 0.05) accompanied by severe lactic acidemia (P < 0.05) in all pigs receiving a high dose of SNP. Administration of the sulfanegen antidote resulted in progressive significant reduction in blood lactate and CN levels with 100% survival (P < 0.05), whereas the placebo-treated pigs deteriorated and did not survive (P < 0.05). NaCN study: NaCN injection resulted in CN toxicity accompanied by severe lactic acidosis and mortality in all the pigs. Sulfanegen sodium reversed this toxicity and prevented mortality in all the pigs treated with this antidote. CONCLUSIONS: CN toxicity can be successfully induced in a juvenile pig model with SNP or NaCN. The prodrug, sulfanegen sodium, is effective in reversing CN toxicity induced by SNP or NaCN.

Giardial lipid rafts share virulence factors with secreted vesicles and participate in parasitic infection in mice.

Giardia lamblia, a protozoan parasite, is a major cause of waterborne infection, worldwide. While the trophozoite form of this parasite induces pathological symptoms in the gut, the cyst form transmits the infection. Since Giardia is a noninvasive parasite, the actual mechanism by which it causes disease remains elusive. We have previously reported that Giardia assembles cholesterol and GM1 glycosphingolipid-enriched lipid rafts (LRs) that participate in encystation and cyst production. To further delineate the role of LRs in pathogenesis, we isolated LRs from Giardia and subjected them to proteomic analysis. Various cellular proteins including potential virulence factors-e.g., giardins, variant surface proteins, arginine deaminases, elongation factors, ornithine carbomyltransferases, and high cysteine-rich membrane proteins-were found to be present in LRs. Since Giardia secretes virulence factors encapsulated in extracellular vesicles (EVs) that induce proinflammatory responses in hosts, EVs released by the parasite were isolated and subjected to nanoparticle tracking and proteomic analysis. Two types of EV-i.e., small vesicles (SVs; <100 nm, exosome-like particles) and large vesicles (LVs; 100-400 nm, microvesicle-like particles)-were identified and found to contain a diverse group of proteins including above potential virulence factors. Although pretreatment of the parasite with two giardial lipid raft (gLR) disruptors, nystatin (27 µM) and oseltamivir (20 µM), altered the expression profiles of virulence factors in LVs and SVs, the effects were more robust in the case of SVs. To examine the potential role of rafts and vesicles in pathogenicity, Giardia-infected mice were treated with oseltamivir (1.5 and 3.0 mg/kg), and the shedding of cysts were monitored. We observed that this drug significantly reduced the parasite load in mice. Taken together, our results suggest that virulence factors partitioning in gLRs, released into the extracellular milieu via SVs and LVs, participate in spread of giardiasis and could be targeted for future drug development.

Exploiting drug repositioning for discovery of a novel HIV combination therapy.

The development of HIV drugs is an expensive and a lengthy process. In this study, we used drug repositioning, a process whereby a drug approved to treat one condition is used to treat a different condition, to identify clinically approved drugs that have anti-HIV activity. The data presented here show that a combination of two clinically approved drugs, decitabine and gemcitabine, reduced HIV infectivity by 73% at concentrations that had minimal antiviral activity when used individually. Decreased infectivity coincided with a significant increase in mutation frequency and a shift in the HIV mutation spectrum. These results indicate that an increased mutational load is the primary antiviral mechanism for inhibiting the generation of infectious progeny virus from provirus. Similar results were seen when decitabine was used in combination with another ribonucleotide reductase inhibitor. Our results suggest that HIV infectivity can be decreased by combining a nucleoside analog that forms noncanonical base pairs with certain ribonucleotide reductase inhibitors. Such drug combinations are relevant since members of these drug classes are used clinically. Our observations support a model in which increased mutation frequency decreases infectivity through lethal mutagenesis.

Distinct Antiretroviral Mechanisms Elicited by a Viral Mutagen.

5-aza-cytidine (5-aza-C) has been shown to be a potent human immunodeficiency virus type 1 (HIV-1) mutagen that induces G-to-C hypermutagenesis by incorporation of the reduced form (i.e., 5-aza-dC, 5-aza-dCTP). Evidence to date suggests that this lethal mutagenesis is the primary antiretroviral mechanism for 5-aza-C. To investigate the breadth of application of 5-aza-C as an antiretroviral mutagen, we have conducted a comparative, parallel analysis of the antiviral mechanism of 5-aza-C between HIV-1 and gammaretroviruses - i.e., murine leukemia virus (MuLV) and feline leukemia virus (FeLV). Intriguingly, in contrast to the hallmark G-to-C hypermutagenesis observed with HIV-1, MuLV and FeLV did not reveal the presence of a significant increase in mutational burden, particularly that of G-to-C transversion mutations. The effect of 5-aza-dCTP on DNA synthesis revealed that while HIV-1 RT was not inhibited by 5-aza-dCTP even at 100 µM, 5-aza-dCTP was incorporated and significantly inhibited MuLV RT, generating pause sites and reducing the fully extended product. 5-aza-dCTP was found to be incorporated into DNA by MuLV RT or HIV-1 RT, but only acted as a non-obligate chain terminator for MuLV RT. This biochemical data provides an independent line of experimental evidence in support of the conclusion that HIV-1 and MuLV have distinct primary mechanisms of antiretroviral action with 5-aza-C. Taken together, our data provides striking evidence that an antiretroviral mutagen can have strong potency via distinct mechanisms of action among closely related viruses, unlinking antiviral activity from antiviral mechanism of action.

Sulfanegen sodium treatment in a rabbit model of sub-lethal cyanide toxicity.

The aim of this study is to investigate the ability of intramuscular and intravenous sulfanegen sodium treatment to reverse cyanide effects in a rabbit model as a potential treatment for mass casualty resulting from cyanide exposure. Cyanide poisoning is a serious chemical threat from accidental or intentional exposures. Current cyanide exposure treatments, including direct binding agents, methemoglobin donors, and sulfur donors, have several limitations. Non-rhodanese mediated sulfur transferase pathways, including 3-mercaptopyruvate sulfurtransferase (3-MPST) catalyze the transfer of sulfur from 3-MP to cyanide, forming pyruvate and less toxic thiocyanate. We developed a water-soluble 3-MP prodrug, 3-mercaptopyruvatedithiane (sulfanegen sodium), with the potential to provide a continuous supply of substrate for CN detoxification. In addition to developing a mass casualty cyanide reversal agent, methods are needed to rapidly and reliably diagnose and monitor cyanide poisoning and reversal. We use non-invasive technology, diffuse optical spectroscopy (DOS) and continuous wave near infrared spectroscopy (CWNIRS) to monitor physiologic changes associated with cyanide exposure and reversal. A total of 35 animals were studied. Sulfanegen sodium was shown to reverse the effects of cyanide exposure on oxyhemoglobin and deoxyhemoglobin rapidly, significantly faster than control animals when administered by intravenous or intramuscular routes. RBC cyanide levels also returned to normal faster following both intramuscular and intravenous sulfanegen sodium treatment than controls. These studies demonstrate the clinical potential for the novel approach of supplying substrate for non-rhodanese mediated sulfur transferase pathways for cyanide detoxification. DOS and CWNIRS demonstrated their usefulness in optimizing the dose of sulfanegen sodium treatment.

Distinct dual antiviral mechanism that enhances hepatitis B virus mutagenesis and reduces viral DNA synthesis.

Reverse transcriptase (RT) is an essential enzyme for the replication of retroviruses and hepadnaviruses. Current therapies do not eliminate the intracellular viral replication intermediate termed covalently closed circular (ccc) DNA, which has enhanced interest in hepatitis B virus (HBV) reverse transcription and cccDNA formation. The HBV cccDNA is generated as a plasmid-like episome in the host cell nucleus from the protein-linked relaxed circular (rc) DNA genome in incoming virions during HBV replication. The creation of the cccDNA via conversion from rcDNA remains not fully understood. Here, we sought to investigate whether viral mutagens can effect HBV replication. In particular, we investigated whether nucleoside analogs that act as viral mutagens with retroviruses could impact hepadnaviral DNA synthesis. We observed that a viral mutagen (e.g., 5-aza-2'-deoxycytidine, 5-aza-dC or 5-azacytidine, 5-aza-C) severely diminished the ability of a HBV vector to express a reporter gene following virus transfer and infection of target cells. As predicted, the treatment of 5-aza-dC or 5-aza-C elevated the HBV rcDNA mutation frequency, primarily by increasing the frequency of G-to-C transversion mutations. A reduction in rcDNA synthesis was also observed. Intriguingly, the cccDNA nick/gap region transcription was diminished by 5-aza-dC, but did not enhance viral mutagenesis. Taken together, our results demonstrate that viral mutagens can impact HBV reverse transcription, and propose a model in which viral mutagens can induce mutagenesis during rcDNA formation and diminish viral DNA synthesis during both rcDNA formation and the conversion of rcDNA to cccDNA.

Phosphonoxins II: diastereoselective synthesis of phosphonic acid analogues of polyoxins.

A diastereoselective synthesis of polyoxin analogues termed phosphonoxin B1 and B2 has been achieved. The key step was sulfinimine-mediated asymmetric formation of ( RS ,2S,3S,4S)-beta-aminophosphonate 3a or (SS ,2R,3S,4S)-beta-aminophosphonate 7 as the major diastereoisomer. A double stereodifferentiation effect was not observed, and the diastereoselectivity is controlled by the absolute configuration of the sulfinyl group.

Nucleobases and corresponding nucleosides display potent antiviral activities against dengue virus possibly through viral lethal mutagenesis.

Dengue virus affects millions of people worldwide each year. To date, there is no drug for the treatment of dengue-associated disease. Nucleosides are effective antivirals and work by inhibiting the accurate replication of the viral genome. Nucleobases offer a cheaper alternative to nucleosides for broad antiviral applications. Metabolic activation of nucleobases involves condensation with 5-phosphoribosyl-1-pyrophosphate to give the corresponding nucleoside-5'-monophosphate. This could provide an alternative to phosphorylation of a nucleoside, a step that is often rate limiting and inefficient in activation of nucleosides. We evaluated more than 30 nucleobases and corresponding nucleosides for their antiviral activity against dengue virus. Five nucleobases and two nucleosides were found to induce potent antiviral effects not previously described. Our studies further revealed that nucleobases were usually more active with a better tissue culture therapeutic index than their corresponding nucleosides. The development of viral lethal mutagenesis, an antiviral approach that takes into account the quasispecies behavior of RNA viruses, represents an exciting prospect not yet studied in the context of dengue replication. Passage of the virus in the presence of the nucleobase 3a (T-1105) and corresponding nucleoside 3b (T-1106), favipiravir derivatives, induced an increase in apparent mutations, indicating lethal mutagenesis as a possible antiviral mechanism. A more concerted and widespread screening of nucleobase libraries is a very promising approach to identify dengue virus inhibitors including those that may act as viral mutagens.

Novel, orally effective cyanide antidotes.

A series of prodrugs of 3-mercaptopyruvate (3-MP), the substrate for the enzyme 3-mercaptopyruvate/cyanide sulfurtransferase (3-MPST) that converts cyanide to the nontoxic thiocyanate, which are highly effective cyanide antidotes, have been developed. These prodrugs of 3-MP are unique in being not only orally bioavailable, but may be administered up to an hour prior to cyanide as a prophylactic agent and are both rapid- or slow-acting when given parenterally.