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Blocking the pyrimidine nucleotide de novo synthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH) results in the cell cycle arrest and/or differentiation of rapidly proliferating cells including activated lymphocytes, cancer cells, or virally infected cells. Emvododstat (PTC299) is an orally bioavailable small molecule that inhibits DHODH. We evaluated the potential for emvododstat to inhibit the progression of acute myeloid leukemia (AML) using several in vitro and in vivo models of the disease. Broad potent activity was demonstrated against multiple AML cell lines, AML blasts cultured ex vivo from patient blood samples, and AML tumor models including patient-derived xenograft models. Emvododstat induced differentiation, cytotoxicity, or both in primary AML patient blasts cultured ex vivo with 8 of 10 samples showing sensitivity. AML cells with diverse driver mutations were sensitive, suggesting the potential of emvododstat for broad therapeutic application. AML cell lines that are not sensitive to emvododstat are likely to be more reliant on the salvage pathway than on de novo synthesis of pyrimidine nucleotides. Pharmacokinetic experiments in rhesus monkeys demonstrated that emvododstat levels rose rapidly after oral administration, peaking about 2 hours post-dosing. This was associated with an increase in the levels of dihydroorotate (DHO), the substrate for DHODH, within 2 hours of dosing indicating that DHODH inhibition is rapid. DHO levels declined as drug levels declined, consistent with the reversibility of DHODH inhibition by emvododstat. These preclinical findings provide a rationale for clinical evaluation of emvododstat in an ongoing Phase 1 study of patients with relapsed/refractory acute leukemias., Competing Interests: Authors AB, LC, BF, CT, JG, JM, BR, WL, JS, AM, SY, RK, JB, KO’K, RSp, EG, SK and MW are or were employed by PTC Therapeutics and have received salary compensation for time, effort, and hold or held financial interest in the company. Author MS was employed by Notable Labs and RSh was employed by InSeption Group. The authors declare that this study received funding from PTC Therapeutics, Inc. The funder had the following involvement with the study: funded the entire study, including all experiments and outside editorial support., (Copyright © 2022 Branstrom, Cao, Furia, Trotta, Santaguida, Graci, Colacino, Ray, Li, Sheedy, Mollin, Yeh, Kong, Sheridan, Baird, O’Keefe, Spiegel, Goodwin, Keating and Weetall.)

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin (HTT) gene. Consequently, the mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin (HTT) protein levels alleviates motor and neuropathological abnormalities. Investigational drugs aim to reduce HTT levels by repressing HTT transcription, stability or translation. These drugs require invasive procedures to reach the central nervous system (CNS) and do not achieve broad CNS distribution. Here, we describe the identification of orally bioavailable small molecules with broad distribution throughout the CNS, which lower HTT expression consistently throughout the CNS and periphery through selective modulation of pre-messenger RNA splicing. These compounds act by promoting the inclusion of a pseudoexon containing a premature termination codon (stop-codon psiExon), leading to HTT mRNA degradation and reduction of HTT levels., (© 2021. The Author(s).)

PTC596 is an investigational small-molecule tubulin-binding agent. Unlike other tubulin-binding agents, PTC596 is orally bioavailable and is not a P-glycoprotein substrate. So as to characterize PTC596 to position the molecule for optimal clinical development, the interactions of PTC596 with tubulin using crystallography, its spectrum of preclinical in vitro anticancer activity, and its pharmacokinetic-pharmacodynamic relationship were investigated for efficacy in multiple preclinical mouse models of leiomyosarcomas and glioblastoma. Using X-ray crystallography, it was determined that PTC596 binds to the colchicine site of tubulin with unique key interactions. PTC596 exhibited broad-spectrum anticancer activity. PTC596 showed efficacy as monotherapy and additive or synergistic efficacy in combinations in mouse models of leiomyosarcomas and glioblastoma. PTC596 demonstrated efficacy in an orthotopic model of glioblastoma under conditions where temozolomide was inactive. In a first-in-human phase I clinical trial in patients with cancer, PTC596 monotherapy drug exposures were compared with those predicted to be efficacious based on mouse models. PTC596 is currently being tested in combination with dacarbazine in a clinical trial in adults with leiomyosarcoma and in combination with radiation in a clinical trial in children with diffuse intrinsic pontine glioma., (©2021 The Authors; Published by the American Association for Cancer Research.)

6β-Hydroxy-21-desacetyl deflazacort (6β-OH-21-desDFZ) is a major circulating but not biologically active metabolite of deflazacort (DFZ). In vitro studies were performed to evaluate cytochrome P450 (CYP)- and transporter-mediated drug interaction potentials of 6β-OH-21-desDFZ. Up to 50 µM, the highest soluble concentration in the test system, 6β-OH-21-desDFZ weakly inhibited (IC 50  > 50 µM) the enzyme activity of CYPs 1A2, 2B6, 2C8, 2C9, and 2D6, while moderately inhibiting CYP2C19 and CYP3A4 with IC 50 values of approximately 50 and 35 μM, respectively. The inhibition was neither time-dependent nor metabolism-based. Incubation of up to 50 µM 6β-OH-21-desDFZ with plated cryopreserved human hepatocytes for 48 h resulted in no meaningful concentration-dependent induction of either mRNA levels or enzyme activity of CYP1A2, CYP2B6, or CYP3A4. In transporter inhibition assays, 6β-OH-21-desDFZ, up to 50 µM, did not show interaction with human OAT1, OAT3, and OCT2 transporters. It weakly inhibited (IC 50  > 50 µM) human MATE1, MATE2-K, and OCT1 transporter activity, and moderately inhibited human MDR1, OATP1B1, and OATP1B3 transporter activity with IC 50 values of 19.81 μM, 37.62 μM, and 42.22 μM, respectively. 14 C-6β-OH-21-desDFZ was biosynthesized using bacterial biotransformation and the subsequent study showed that 6β-OH-21-desDFZ was not a substrate for human BCRP, MDR1, MATE1, MATE2-K, OAT1, OATP1B1, OATP1B3, and OCT2 transporters, but appeared to be an in vitro substrate for the human OAT3 uptake transporter. At plasma concentrations of 6β-OH-21-desDFZ seen in the clinic, CYP- and transporter-mediated drug-drug interactions are not expected following administration of a therapeutic dose of DFZ in Duchenne muscular dystrophy (DMD) patients., (© 2021 PTC Therapeutics, Inc. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS-COV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti-COVID-19 potential of PTC299, an orally bioavailable compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine nucleotide biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS-COV-2 replication (EC 50 range, 2.0-31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17 F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19., (Copyright © 2020. Published by Elsevier B.V.)

Ataluren promotes ribosomal readthrough of premature termination codons in mRNA which result from nonsense mutations. In vitro studies were performed to characterize the metabolism and enzyme kinetics of ataluren and its interaction potential with CYP enzymes. Incubation of [ 14 C]-ataluren with human liver microsomes indicated that the major metabolic pathway for ataluren is via direct glucuronidation and that the drug is not metabolized via cytochrome P450 (CYP). Glucuronidation was also observed in the incubation in human intestinal and kidney microsomes, but not in human pulmonary microsomes. UGT1A9 was found to be the major uridine diphosphate glucuronosyltransferase (UGT) responsible for ataluren glucuronidation in the liver and kidney microsomes. Enzyme kinetic analysis of the formation of ataluren acyl glucuronide, performed in human liver, kidney, and intestinal microsomes and recombinant human UGT1A9, found that increasing bovine serum albumin (BSA) levels enhanced the glucuronidation Michaelis-Menten constant (K m ) and ataluren protein binding but had a minimal effect on maximum velocity (V max ) of glucuronidation. Due to the decreased unbound Michaelis-Menten constant (K m,u ), the ataluren unbound intrinsic clearance (CL int,u ) increased for all experimental systems and BSA concentrations. Human kidney microsomes were about 3.7-fold more active than human liver microsomes, in terms of CL int,u /mg protein, indicating that the kidney is also a key organ for the metabolism and disposition of ataluren in humans. Ataluren showed no or little potential to inhibit or induce most of the CYP enzymes., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

PTC299 was identified as an inhibitor of VEGFA mRNA translation in a phenotypic screen and evaluated in the clinic for treatment of solid tumors. To guide precision cancer treatment, we performed extensive biological characterization of the activity of PTC299 and demonstrated that inhibition of VEGF production and cell proliferation by PTC299 is linked to a decrease in uridine nucleotides by targeting dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis. Unlike previously reported DHODH inhibitors that were identified using in vitro enzyme assays, PTC299 is a more potent inhibitor of DHODH in isolated mitochondria suggesting that mitochondrial membrane lipid engagement in the DHODH conformation in situ is required for its optimal activity. PTC299 has broad and potent activity against hematologic cancer cells in preclinical models, reflecting a reduced pyrimidine nucleotide salvage pathway in leukemia cells. Archived serum samples from patients treated with PTC299 demonstrated increased levels of dihydroorotate, the substrate of DHODH, indicating target engagement in patients. PTC299 has advantages over previously reported DHODH inhibitors, including greater potency, good oral bioavailability, and lack of off-target kinase inhibition and myelosuppression, and thus may be useful for the targeted treatment of hematologic malignancies., (©2018 American Association for Cancer Research.)

Nonsense mutations, resulting in a premature stop codon in the open reading frame of mRNAs are responsible for thousands of inherited diseases. Readthrough of premature stop codons by small molecule drugs has emerged as a promising therapeutic approach to treat disorders resulting from premature termination of translation. The aminoglycoside antibiotics are a class of molecule known to promote readthrough at premature termination codons. Gentamicin consists of a mixture of major and minor aminoglycoside components. Here, we investigated the readthrough activities of the individual components and show that each of the four major gentamicin complex components representing 92-99% of the complex each had similar potency and activity to that of the complex itself. In contrast, a minor component (gentamicin X2) was found to be the most potent and active readthrough component in the gentamicin complex. The known oto- and nephrotoxicity associated with aminoglycosides preclude long-term use as readthrough agents. Thus, we evaluated the components of the gentamicin complex as well as the so-called "designer" aminoglycoside, NB124, for in vitro and in vivo safety. In cells, we observed that gentamicin X2 had a safety/readthrough ratio (cytotoxicity/readthrough potency) superior to that of gentamicin, G418 or NB124. In rodents, we observed that gentamicin X2 showed a safety profile that was superior to G418 overall including reduced nephrotoxicity. These results support further investigation of gentamicin X2 as a therapeutic readthrough agent., Competing Interests: All authors are employees and stock holders of PTC Therapeutics. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Despite advances in antiretroviral therapy, HIV-1 infection remains incurable in patients and continues to present a significant public health burden worldwide. While a number of factors contribute to persistent HIV-1 infection in patients, the presence of a stable, long-lived reservoir of latent provirus represents a significant hurdle in realizing an effective cure. One potential strategy to eliminate HIV-1 reservoirs in patients is reactivation of latent provirus with latency reversing agents in combination with antiretroviral therapy, a strategy termed "shock and kill". This strategy has shown limited clinical effectiveness thus far, potentially due to limitations of the few therapeutics currently available. We have identified a novel class of benzazole compounds effective at inducing HIV-1 expression in several cellular models. These compounds do not act via histone deacetylase inhibition or T cell activation, and show specificity in activating HIV-1 in vitro. Initial exploration of structure-activity relationships and pharmaceutical properties indicates that these compounds represent a potential scaffold for development of more potent HIV-1 latency reversing agents.

Nonsense mutations resulting in a premature stop codon in an open reading frame occur in critical tumor suppressor genes in a large number of the most common forms of cancers and are known to cause or contribute to the progression of disease. Low molecular weight compounds that induce readthrough of nonsense mutations offer a new means of treating patients with genetic disorders or cancers resulting from nonsense mutations. We have identified the nucleoside analog clitocine as a potent and efficacious suppressor of nonsense mutations. We determined that incorporation of clitocine into RNA during transcription is a prerequisite for its readthrough activity; the presence of clitocine in the third position of a premature stop codon directly induces readthrough. We demonstrate that clitocine can induce the production of p53 protein in cells harboring p53 nonsense-mutated alleles. In these cells, clitocine restored production of full-length and functional p53 as evidenced by induced transcriptional activation of downstream p53 target genes, progression of cells into apoptosis, and impeded growth of nonsense-containing human ovarian cancer tumors in xenograft tumor models. Thus, clitocine induces readthrough of nonsense mutations by a previously undescribed mechanism and represents a novel therapeutic modality to treat cancers and genetic diseases caused by nonsense mutations., (© 2017 Friesen et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal "homeostatic" state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5' and 3' untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies., Competing Interests: We have the following interests: The authors are employed by and received monetary compensation from PTC Therapeutics, Inc. Liangxian Cao, Marla Weetall, Jenelle Bombard (former employee), Hongyan Qi (former employee), Tamil Arasu (former employee), William Lennox, Jean Hedrick, Josephine Sheedy, Nicole Risher, Panayiota Trifillis, Christopher Trotta, Young-Choon Moon, John Babiak, Neil G Almstead, Joseph M Colacino, Thomas W Davis (former employee) and Stuart W Peltz are or were employed by and hold or held financial interests in PTC Therapeutics, Inc. PTC Therapeutics hold the patents pertaining to the results presented in this paper as below: Liangxian Cao and Panayiota Trifillis, Methods for Identifying Compounds that Modulate Untranslated Region-Based Regulation, CA2514184 and US8460864. Christopher R Trotta and Liangxian Cao, Methods and Agents for Screening for Compounds Capable of Modulating VEGF Expression, CA2567111, EP2400038 and US8426194. Liangxian Cao; William Lennox; Hongyan Qi; Young-Choon Moon and Nadarajan Tamilarasu, Tetra-Cyclic Carboline Derivatives Useful in the Inhibition of Angiogenesis, MX286760 and US8940896. Young-Choon Moon; William Lennox and Hongyan Qi, Tetrahydrocarbazoles as Active Agents for Inhibiting VEGF Production by Translational Control, US8946444 and US9271960. There are no additional patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

PTC725 is a small molecule NS4B-targeting inhibitor of hepatitis C virus (HCV) genotype (gt) 1 RNA replication that lacks activity against HCV gt2. We analyzed the Los Alamos HCV sequence database to predict susceptible/resistant HCV gt's according to the prevalence of known resistance-conferring amino acids in the NS4B protein. Our analysis predicted that HCV gt3 would be highly susceptible to the activity of PTC725. Indeed, PTC725 was shown to be active against a gt3 subgenomic replicon with a 50% effective concentration of ∼5 nM. De novo resistance selection identified mutations encoding amino acid substitutions mapping to the first predicted transmembrane region of NS4B, a finding consistent with results for PTC725 and other NS4B-targeting compounds against HCV gt1. This is the first report of the activity of an NS4B targeting compound against HCV gt3. In addition, we have identified previously unreported amino acid substitutions selected by PTC725 treatment which further demonstrate that these compounds target the NS4B first transmembrane region., (Copyright © 2016 Graci et al.)

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA., (Copyright © 2014, American Association for the Advancement of Science.)

A novel, potent, and orally bioavailable inhibitor of hepatitis C RNA replication targeting NS4B, compound 4t (PTC725), has been identified through chemical optimization of the 6-(indol-2-yl)pyridine-3-sulfonamide 2 to improve DMPK and safety properties. The focus of the SAR investigations has been to identify the optimal combination of substituents at the indole N-1, C-5, and C-6 positions and the sulfonamide group to limit the potential for in vivo oxidative metabolism and to achieve an acceptable pharmacokinetic profile. Compound 4t has excellent potency against the HCV 1b replicon, with an EC50 = 2 nM and a selectivity index of >5000 with respect to cellular GAPDH. Compound 4t has an overall favorable pharmacokinetic profile with oral bioavailability values of 62%, 78%, and 18% in rats, dogs, and monkeys, respectively, as well as favorable tissue distribution properties with a liver to plasma exposure ratio of 25 in rats.

While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC50) of 1.7 nM and an EC90 of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.

Professor Erik De Clercq, recent recipient of the International Society for Antiviral Research (ISAR) 'Outstanding Contributions to the Society Award', recounts 25 years of antiviral research collaborating with his colleagues and friends in Japan.

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.

The present study investigates some aspects of the digestive biology and physiological energetics of the Florida lancelet, Branchiostoma floridae. Florida lancelets are able to remove 47.2-56.9% of the energy from a diet of mixed algae. The respiration rate is 0.100mL O(2) (STPD) h(-1) g(-1) (wet), which estimates a metabolic rate of 0.248 J h(-1), at an average body mass of 0.125 g (wet). Published values of the chlorophyll a concentration in its natural habitat indicate that a 125 mg lancelet would need to filter 0.018-0.031 L h(-1) to remove sufficient food to support its resting metabolism. The filtration rate of lancelets has been reported as 0.138 L h(-1), indicating that the actual filtration rate is 4-7 times greater than the filtration rate needed to meet resting metabolic demands. It appears that lancelets have the potential to be raised in aquaculture, because their absorption efficiency and respiration rate are comparable to suspension-feeding invertebrates that have been successfully aquacultured.

HIV type-1 (HIV-1) can establish a state of latency in infected patients, most notably in resting CD4(+) T-cells. This long-lived reservoir allows for rapid re-emergence of viraemia upon cessation of highly active antiretroviral therapy, even after extensive and seemingly effective treatment. Successful depletion of such latent reservoirs is probably essential to 'cure' HIV-1 infection and will require therapeutic agents that can specifically and efficiently act on cells harbouring latent HIV-1 provirus. The mechanisms underlying HIV-1 latency are not well characterized, and it is becoming clear that numerous factors, both cell- and virus-derived, are involved in the maintenance of proviral latency. The interplay of these various factors in the context of viral reactivation is still poorly understood. In this article, we review the current knowledge regarding the mechanisms underlying maintenance of HIV-1 latency, both transcriptional and post-transcriptional, with a focus on potential targets that might be exploited to therapeutically purge latent proviral reservoirs from infected patients.

Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.

The burrowing brittle star Hemipholis elongata (Say) possesses hemoglobin-containing coelomocytes (RBCs) in its water vascular system. The RBCs, which circulate between the arms and body, are thought to play a role in oxygen transport. The hemoglobin of adult animals has a moderate affinity for oxygen (P(50) = 11.4 mm Hg at pH 8, 20 degrees C, measured in cellulo) and exhibits cooperativity (Hill coefficient > 1.7). The hemoglobin of juveniles has a higher affinity (P(50) = 2.3 mmHg at pH 8.0, 20 degrees C) and also exhibits cooperativity. The oxygen-binding properties of the hemoglobin are relatively insensitive to pH, temperature, and hydrogen sulfide. Adult hemoglobin is a heterogeneous mixture composed of three major fractions. The combined results of electrospray mass spectrometry and oxygen-binding experiments performed on purified fractions indicate that the native hemoglobin is in the form of homopolymers. A partial amino acid sequence (about 40 amino acids) of adult hemoglobin reveals little homology with holothurian hemoglobins.

The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of approximately 22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 A. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.

To examine the role of the mitochondrial polymerase (Pol gamma) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU), beta-L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol gamma holoenzyme in single turnover kinetic studies to measure K(d) (K(m)) and k(pol) (k(cat)) to estimate the specificity constant (k(cat)/K(m)) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2',3'-didehydro-2',3'-dideoxythymidine (d4T) >> (+)3TC >> (-)3TC > PMPA > azidothymidine (AZT) >> Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC approximately equal to (-)3TC > CBV > AZT > PMPA approximately equal to d4T >> ddA (ddI) >> ddC. Removal of ddC was too slow to measure (<0.00002 s(-1)). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s(-1)) faster than it is removed (0.06 s(-1)) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.

We have recently shown that the replication of an HCV-poliovirus (PV) chimera that is dependent upon the hepatitis C virus (HCV) 5' untranslated region (UTR) can be inhibited by treatment with ribozymes targeting HCV RNA. To determine the antiviral effects of anti-HCV ribozyme treatment in combination with type 1 interferon (IFN), we analysed the replication of this HCV-PV chimera in HeLa cells treated with anti-HCV ribozyme and/or IFN-alpha2a, IFN-alpha2b, or consensus IFN. The anti-HCV ribozyme, or any of the IFNs alone have significant inhibitory effects on HCV-PV replication compared to control treatment (> or = 85%, P < 0.01). The maximal inhibition due to IFN treatment (94%, P < 0.01) was achieved with > or = 50 U/ml for either IFN-alpha2a or IFN-alpha2b compared to control treatment. A similar level of inhibition in viral replication could be achieved with a 5-fold lower dose of IFN if ribozyme targeting the HCV 5' UTR was given in combination. For consensus IFN, the dose could be reduced by > 12.5-fold if ribozyme targeting the HCV 5' UTR was given in combination. Conversely, the dose of ribozyme could be reduced 3-fold if given in combination with any of the IFN preparations. Moreover, treatment with low doses (1-25 U/mL) of IFN-alpha2a, IFN-alpha2b, or consensus IFN in combination with anti-HCV ribozyme resulted in > 98% inhibition of HCV-PV replication compared to control treatment (P < 0.01). These results demonstrate that IFN and ribozyme each have a beneficial antiviral effect that is augmented when given in combination.

A safe and effective vaccine for hepatitis B virus (HBV) has been available for nearly twenty years and currently campaigns to provide universal vaccination in developing countries are underway. Nevertheless, chronic HBV infection remains a leading cause of chronic hepatitis worldwide and there is a strong need for safe and effective antiviral therapies. Attempts to identify and develop antiviral agents to treat chronic HBV infection remains focused on nucleoside analogs such as 3TC (lamivudine), adefovir dipivoxil, (bis-POMPMEA), and others. However, advances in our understanding of the molecular biology of HBV and the development of new assays for HBV polymerase activity, such as the reconstitution of active HBV polymerase in vitro, should facilitate large screening efforts for non-nucleoside reverse transcriptase inhibitors. Recent advances have furthered our understanding of clinical resistance to lamivudine, have provided new approaches to treatment, and have offered new perspectives on the major challenges to the identification and development of antiviral agents for chronic HBV infection. Here, in an update to our previous review article that appeared in this series [59a], we focus on recent advances that have occurred in the areas of virus structure and replication, in vitro viral polymerase assays, cell culture systems, and animal models.

The burrowing brittlestar Hemipholis elongata (Say) maintains a constant M(O2)of 3.79+/-1.47 micromol O(2) g(-1) h(-1) (for 0.2-0.3 g animals, mean+/-S.D., n=7), measured in the burrow, over a broad range of PO(2). Below the critical PO(2) of 37 mm Hg, M(O(2)) becomes dependent on the oxygen tension. M(O2) is a function of the size of H. elongata; the scaling exponent is 0. 83 and is similar to those reported for other echinoderms. The M(O2) of H. elongata is unaffected by removal from the burrow, by hypercapnia, by exposure to hydrogen sulfide, or by temperature change in the range from 20 to 32 degrees C. The relative insensitivity of H. elongata to these factors may be an adaptation to life in the highly variable estuarine and tidal creek environments where the animals are frequently found.

Influenza A and B viruses belong to the Orthomyxoviridae family of viruses. These viruses are responsible for severe morbidity and significant excess mortality each year. Infection with influenza viruses usually leads to respiratory involvement and can result in pneumonia and secondary bacterial infections. Vaccine approaches to the prophylaxis of influenza virus infections have been problematic owing to the ability of these viruses to undergo antigenic shift by exchanging genomic segments or by undergoing antigenic drift, consisting of point mutations in the haemagglutinin (HA) and neuraminidase (NA) genes as a result of an error-prone viral polymerase. Historically, antiviral approaches for the therapy of both influenza A and B viruses have been largely unsuccessful until the elucidation of the X-ray crystallographic structure of the viral NA, which has permitted structure-based drug design of inhibitors of this enzyme. In addition, recent advances in the elucidation of the structure and complex function of influenza HA have resulted in the discovery of a number of diverse compounds that target this viral protein. This review article will focus largely on newer antiviral agents including those that inhibit the influenza virus NA and HA. Other novel approaches that have entered clinical trials or been considered for their clinical utility will be mentioned.

Entry of influenza virus into the host cell is dependent on the fusion of the viral envelope with the endosomal membrane and is mediated by a low-pH-induced change of the viral hemagglutinin (HA) to a conformation that is fusogenic. A compound related to podocarpic acid (180299) was identified that inhibits multicycle replication of influenza A/Kawasaki/86 (H1N1) virus in culture. Treatment of Madin-Darby canine kidney (MDCK) cells with 180299 at 1 h before infection resulted in the inhibition of viral protein synthesis. Addition of 20 microgram of 180299/ml at 1 h p.i. had no effect, indicating that 180299 affects an early step of the influenza viral replication cycle. Genetic analysis of reassortants between sensitive and resistant viruses demonstrated that hemagglutinin (HA) conferred the 180299-resistant (180299(r)) phenotype. Twelve independent isolates of influenza A/Kawasaki/86 were selected for resistance to 180299, and sequence analysis revealed that each of these viruses contained amino acid substitutions in the HA. These mutations are dispersed throughout the HA primary amino acid sequence and cluster in one of two regions: the interface between HA1 and HA2 and in a region near the fusion domain of HA2. When compared with the parent virus, the pH-of-inactivation of the resistant mutants was increased by 0.3 to 0.6 pH unit, suggesting that the mutant HAs undergo the conformational change at an elevated pH. Fusion of human erythrocytes to MDCK cells infected with parent influenza A/Kawasaki/86 was inhibited by 180299 (0.1-10 microgram/ml) in a concentration-dependent manner, whereas fusion of erythrocytes to MDCK cells infected with 180299(r) mutants was not affected. These results suggest that 180299 interacts with the neutral pH conformation of influenza A HA and prevents the low-pH-induced change of HA to its fusogenic conformation., (Copyright 1998 Academic Press.)

Hemoglobin (Hb) occurs in circulating red blood cells, neural tissue, and body wall muscle tissue of the nemertean worm, Cerebratulus lacteus. The neural and body wall tissue each express single major Hb components for which the amino acid sequences have been deduced from cDNA and genomic DNA. These 109-residue globins form the smallest stable Hbs known. The globin genes have three exons and two introns with splice sites in the highly conserved positions of most globin genes. Alignment of the sequences with those of other globins indicates that the A, B, and H helices are about one-half the typical length. Phylogenetic analysis indicates that shortening results in a small tendency of globins to group together regardless of their actual relationships. The neural and body wall Hbs in situ are half-saturated with O2 at 2.9 and 4.1 torr, respectively. The Hill coefficient for the neural Hb in situ, approximately 2.9, suggests that the neural Hb self-associates in the deoxy state at least to tetramers at the 2-3 mM (heme) concentration estimated in the cells. The Hb must dissociate upon oxygenation and dilution because the weight-average molecular mass of the HbO2 in vitro is only about 18 kDa at 2-3 microM heme concentration. Calculations suggest that the Hb can function as an O2 store capable of extending neuronal activity in an anoxic environment for 5-30 min.

Woodchucks were used to study the antiviral activity and toxicity of fialuridine (FIAU; 1,-2'deoxy-2'fluoro-1-beta-D-arabinofuranosyl-5-iodo-uracil). In an initial experiment, groups of six chronic woodchuck hepatitis virus (WHV) carrier woodchucks received daily doses of FIAU by intraperitoneal injection for 4 weeks. At 0.3 mg/kg/d, the antiviral effect was equivocal, but at 1.5 mg/kg/d, FIAU had significant antiviral activity. No evidence of drug toxicity was observed during the 4-week period of treatment or during posttreatment follow-up. In a second experiment, groups of nine WHV carriers or uninfected woodchucks were given 1.5 mg/kg/d of FIAU orally for 12 weeks, and the results compared with placebo-treated controls. After 4 weeks, the serum WHV-DNA concentration in the FIAU-treated carrier group was two to three logs lower than that in the placebo-treated group. After 12 weeks of FIAU treatment, serum WHV DNA was not detectable by conventional dot-blot analysis, hepatic WHV-DNA replicative intermediates (RI) had decreased 100-fold, and hepatic expression of WHV core antigen was remarkably decreased. No evidence of toxicity was observed after 4 weeks, but, after 6 to 7 weeks, food intake decreased and, after 8 weeks, the mean body weights of woodchucks treated with FIAU were significantly lower than controls. Anorexia, weight loss, muscle wasting, and lethargy became progressively severe, and all FIAU-treated woodchucks died or were euthanized 78 to 111 days after treatment began. Hepatic insufficiency (hyperbilirubinemia, decreased serum fibrinogen, elevated prothrombin time), lactic acidosis, and hepatic steatosis were characteristic findings in the final stages of FIAU toxicity in woodchucks. The syndrome of delayed toxicity in woodchucks was similar to that observed previously in humans treated with FIAU, suggesting that the woodchuck should be valuable in future investigations of the molecular mechanisms of FIAU toxicity in vivo and for preclinical toxicological evaluation of other nucleoside analogs before use in patients.

The synthesis and antiviral evaluation of 21 prodrugs of 1-[2',3'-dideoxy-3'-C-(hydroxymethyl)-beta-D-erythropentofuranosyl ] cytosine 1 is reported. Cytosine N4-imine analogues were prepared by condensation of 1 with selected formamide dimethyl acetals. Amino acid substituted prodrugs were prepared from 1 or imine prodrug 2 by coupling with either N-tert-butoxycarbonyl (t-Boc)-L-valine or N-t-Boc-L- phenylalanine in the presence of dicyclohexycarbodiimide (DCC) and 4-dimethylaminopyridine (4-DMAP). Deprotection of the t-Boc protecting group was achieved with trifluoroacetic acid (TFAA) in methylene chloride. Cytosine N4-amide analogues were prepared by reaction of 1 with appropriate anhydrides in aqueous dioxane. Triacylated analogue 22 was prepared by reaction of 1 with four equivalents of benzoyl chloride in pyridine. Prodrugs were evaluated for activity against duck hepatitis B virus, herpes simplex virus types 1 and 2, human cytomegalovirus, and human immunodeficiency virus. A number of analogues were found comparable in activity to 1 with the cytosine N4-imine series more active than the amino acid substituted and cytosine N4-amide prodrugs. Slight to moderate cellular toxicity was observed with some analogues.

Hepatitis B virus (HBV) is the leading cause of chronic hepatitis throughout the world. Notwithstanding the availability of a safe and effective vaccine, the world prevalence of HBV has not declined significantly, thus resulting in the need for a selective antiviral agent. HBV is a small, partially double-stranded DNA virus which replicates through an RNA intermediate. Most efforts to develop anti-HBV agents have been targeted to the viral DNA polymerase which possesses reverse transcriptase activity. Currently, the most promising anti-HBV agents are nucleoside analogs which interfere with viral DNA replication. Although earlier nucleoside analogs such as vidarabine (ara-A) and fialuridine (FIAU) have displayed unacceptable toxicities, newer analogs such as lamivudine (3TC), bis-POM PMEA (GS-840), lobucavir, and BMS-200,475 have demonstrated clinical utility. In particular, the use of lamivudine has generated considerable interest in the development of other L-enantiomeric nucleoside analogs for use against HBV. Here, we provide an overview of HBV structure and replication strategy and discuss the use of cell culture systems, in vitro viral polymerase systems, and animal models to identify and evaluate anti-HBV agents. We also discuss the various classes of nucleoside analogs in terms of structure, mechanism of action, status in clinical development, ability to select for resistant HBV variants, and use in combination therapies. Finally, we present a discussion of novel antiviral approaches, including antisense and gene therapy, and address the various challenges to successful anti-HBV chemotherapeutic intervention.

A single change (E119G) in the influenza A virus N9 neuraminidase (NA) results in resistance of the enzyme to the NA inhibitor 4-Guanidino-Neu5Ac2en (4-GuDANA). This change causes a salt link between Glu119, which sits in a pocket in the bottom of the active site of the enzyme, and the 4-guanidinium moiety of the inhibitor to be lost. NA "heads" of the resistant enzyme produced only a few small crystals under conditions in which the wild-type enzyme readily formed large crystals. These small crystals were of sufficient quality to yield X-ray crystallographic data which confirmed the E119G change and demonstrated the presence of electron density representing either a strong structural-water molecule or an anionic species in place of the glutamate carboxylate. NA heads of the resistant enzyme also have greatly reduced NA activity per milligram of total protein. We have now found that the mutant NA heads consist predominantly of monomers with a few dimers and tetramers, as determined by electron microscopic analysis of the protein. The low level of enzymatic activity as well as the small number of crystals obtained were probably from the few tetramers remaining intact in the preparation. The purified wild-type and 4-GuDANA-resistant enzymes were treated with the homobifunctional NHS-ester cross linker, DTSSP. SDS-PAGE analysis of the treated enzymes clearly revealed cross-linked dimers of the wild-type enzyme. In contrast, only a small proportion of the 4-GuDANA-resistant neuraminidase was cross-linked. An examination of the known X-ray crystallographic structure of the wild-type NA reveals a salt bridge between Glu119 and Arg156 of the same monomer. Arg156 is a conserved amino acid that is situated at the interface between monomers, and a salt link between this amino acid and Glu119 may contribute to the stability of enzyme tetramers. It is suggested that the E119G alteration in the 4-GuDANA-resistant NA leads to the abrogation of this interaction and thus to the instability of the NA tetramers., (Copyright 1997 Academic Press.)

The reassortant influenza viruses, A/NWS-G70c with N9 neuraminidase (NA) and B/HK/8/73 (HG) with B/Lee/40 NA, were selected for resistance to 4-guanidino-Neu5Ac2en (4-GuDANA) by passaging the virus in stepwise increases in the concentration of 4-GuDANA. In the NA of resistant viruses, the absolutely conserved Glu 119, which lies in a pocket beneath the active site of the enzyme and interacts with the guanidinium moiety of 4-GuDANA, was changed to Gly. The mutant NA was >200-fold more resistant to 4-GuDANA than was the wild-type enzyme. During 72 h in cell culture, resistant A and B viruses displayed much less NA activity than did wild-type viruses but did undergo multicycle replication. While emergence of resistance to 4-GuDANA has not been observed in vivo, these results demonstrate that the development of resistance is possible and can be mediated by a single amino acid change in the active site of the viral NA.

Fialuridine (FIAU) is a thymidine analog effective against hepatitis B virus. Toxicity associated with FIAU treatment included clinical signs consistent with mitochondrial dysfunction, including severe lactic acidosis. To understand further the mechanism of FIAU toxicity, we examined the effect of FIAU on DNA synthesis in mitochondria. Mitochondria isolated from livers of naive rats were treated in vitro with concentrations of FIAU or FIAU triphosphate (FIAU-TP) ranging from 0.1 to 200 microM. A 14 or 32% decrease in mitochondrial DNA synthesis compared to controls was observed when isolated mitochondria were treated with 25 microM FIAU or FIAU-TP, respectively. Since it is thought that nucleosides must be phosphorylated to inhibit DNA polymerase, studies were conducted to determine whether isolated rat mitochondria could phosphorylate FIAU. Results using lanthanum chloride precipitation and HPLC analysis showed that enzymes present in a mitochondrial lysate were capable of phosphorylating FIAU to form FIAU monophosphate.

The thymidine analog, 1-(2-deoxy-2-fluoro-beta-D-arabino-furanosyl)-5-iodouracil (FIAU), is incorporated into DNA in cell culture and in vivo. To investigate the effect of incorporation of FIAU into DNA on the binding of transcription factors, oligonucleotide duplexes which bind specifically to activator protein 1 (AP-1) or to TFIID were synthesized and binding of these oligonucleotides to their respective proteins was studied using gel-shift analysis. When thymidine at position -3, -1, 1 or 7 (relative to the first thymidine of the core binding sequence) was replaced with FIAU, binding to AP-1 was approximately 82, 28, 86 and 51%, respectively, of the binding to the non-substituted oligonucleotide to AP-1. When thymidine at position 3 or 5 (each adjacent to the center of dyad symmetry) was replaced with FIAU, binding to AP-1 was abrogated. Oligonucleotides containing FIAU at positions -1, 3 or 5, were much less able to compete with radiolabeled wild-type oligonucleotides for binding to AP-1. In contrast, the presence of FIAU, depending on its location, resulted in the increased binding of TFIID to its consensus target DNA sequence. These results indicate that incorporation of FIAU into DNA may induce local conformational changes resulting in the altered ability of transcriptional factors to bind to their cognate DNA sequences. Additional studies demonstrated that the presence of FIAU at a position 5' to the cleavage site in the consensus sequence T*TAA (where * is the cleavage site) inhibited restriction of the oligomeric duplex by MseI.

Fialuridine (FIAU), a thymidine nucleoside analogue with anti-hepatitis B virus activity, showed clinical toxicity consistent with mitochondrial dysfunction. In vitro methods were used to understand further this toxicity. Using a sensitive and specific radioimmunoassay, FIAU was found to be present in nuclear DNA of human hepatoblastoma cells incubated for 6 days in 10 or 50 n M drug, at a level of 1 residue per 63 or 39 thymidines, respectively, and was present in mitochondrial DNA at a level of 1 residue per 2139 or 1696 thymidines, respectively. Human hepatoblastoma cells were incubated for 6 days in increasing concentrations of FIAU or, for comparative purposes, the nucleoside analogue dideoxycytidine (ddC), after which time the cells were examined by electron microscopy. At 10 mum and higher concentrations, both compounds induced morphological changes in the ultrastructure of mitochondria characterized by marked mitochondrial swelling, loss of internal cristae and dissolution of the internal matrix. These results, considered along with previously published studies, indicate that FIAU has deleterious effects in vitro on mitochondrial function and structure that occur relatively quickly but without an apparent decrease in the abundance of mitochondrial DNA.

The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.

Fialuridine (FIAU) is a thymidine nucleoside analog with activity against various herpesviruses and hepatitis B virus (HBV) in vitro and in vivo. In a clinical evaluation for its use as a treatment for chronic HBV infection, long term (HBV) in vitro and in vivo. In a clinical evaluation for its term oral administration of FIAU resulted in severe multi-organ toxicity characterized by a delayed onset and refractory lactic acidosis. These clinical manifestations led to the hypothesis that the toxicity of FIAU was mediated through mitochondrial dysfunction, possibly as a result of the inhibition of mitochondrial DNA polymerase gamma and/or incorporation of FIAU into mitochondrial DNA. In addition to describing the anti-HBV activity of FIAU, this review discusses results from in vitro experiments carried out by various laboratories in an effort to evaluate and understand more fully the mitochondrial toxicity of FIAU.

We report the selection and characterization of influenza A/NWS-G70c and B/HK/8/73 (HG) viruses which are resistant to the potent influenza neuraminidase inhibitor, 4-guanidino-Neu5Ac2en. Viruses were selected which replicated in MDCK cells in the presence of 20 micrograms/ml inhibitor. The neuraminidase of resistant viruses was > 200-fold more resistant to 4-guanidino-Neu5Ac2en than was the neuraminidase of the parent viruses. Although amounts of neuraminidase protein were similar in resistant and parent viruses, the enzyme activity of the resistant neuraminidase heads was reduced by > 95% for the substrates used. Relative to parent viruses, the resistant viruses replicated to equal or greater titers in tissue culture and in embryonated chicken eggs. Sequence analysis revealed a single nucleotide mutation in the neuraminidase gene of each virus resulting in the change of the conserved Glu 119 (which lies in a pocket beneath the active site of the enzyme) to Gly thus eliminating an electrostatic interaction with the C-4 guanidinium moiety of the inhibitor. Mutations (Asn-->Ser) at amino acids 145 and 150 were also found in the hemagglutinin gene of the B/HK/8/73 (HG) virus resistant to 4-guanidino-Neu5Ac2en. No changes were found in the hemagglutinin gene of the resistant A/NWS-G70c virus.

To identify the minimal structural elements necessary for biological activity, the rigid tricyclic nucleus of the known human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor tetrahydroimidazobenzodiazepinthione was subjected to systematic bond disconnection to obtain simpler structures. A rational selection and testing of modeled analogs containing these potential pharmacophoric moieties led to the discovery of a new series of nonnucleoside inhibitors of RT. The lead compound of this new PETT series of nonnucleoside RT inhibitors, N-(2-phenylethyl)-N'-(2-thiazolyl)thiourea (LY73497), was found to inhibit HIV-1 but not HIV-2 or simian immunodeficiency virus in cell culture at micromolar concentrations. This derivative was also found to inhibit HIV-1 RT. Through an integrated effort involving synthesis and molecular modeling, compounds with nanomolar potency against HIV-1 in cell culture were developed. In these studies, LY300046-HCl was identified as a potent nonnucleoside inhibitor of HIV-1 RT possessing favorable pharmacokinetic properties.

Fialuridine [FIAU, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- iodouridine] was used in clinical trials for chronic hepatitis B virus infection and was extremely toxic. Evidence suggested targets of FIAU toxicity included mitochondria, but toxic mechanisms were unclear. Since FIAU is a thymidine analog, we reasoned that triphosphorylated FIAU (FIAUTP) could be incorporated into mitochondrial DNA by DNA pol-gamma and into genomic DNA by DNA polymerases alpha, beta, delta, and epsilon. All five purified mammalian DNA polymerases incorporated FIAUMP into the nascent DNA chain during in vitro DNA synthesis. When FIAUTP was substituted for dTTP, oligonucleotide products were generated efficiently by DNA pol-gamma and were similar to those generated in the presence of the four normal dNTPs. In contrast, oligonucleotide products generated by the four nuclear DNA polymerases in the presence of FIAUTP were significantly reduced in length relative to those generated in the presence of dTTP. In parallel kinetic assays, FIAUTP competitively inhibited the accumulation of radiolabeled dTTP into DNA by DNA pol-gamma. The Ki with DNA pol-gamma was 0.04 microM, the lowest Ki among the mammalian DNA polymerases. Competition between FIAUTP and dTTP and the relative ease of accumulation of FIAUMP in mitochondrial DNA by DNA pol-gamma in vitro together may relate to clinical FIAU toxicity.

Hepadnaviruses employ a unique mechanism for the initiation of RNA-directed DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nucleotide analogs. By using cell-free expression of duck hepatitis B virus (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992), the 5'-triphosphate of the thymidine analog fialuridine (FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphosphate of FIAU (FIAU-TP) was nearly complete at a concentration of 10 microM. The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidine triphosphate, known inhibitors of DHBV endogenous DNA polymerase, did not affect substantially the synthesis of primer DNA. Alternate substrate analysis suggested that FIAU is incorporated efficiently into nascent primer DNA as an analog of thymidine. Using site-directed mutagenesis to construct a mutant RNA template yielding a primer with the sequence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorporation of TMP, had no effect on that of dAMP, and decreased markedly the incorporation of dCMP. These results show that the synthesis of full-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideoxynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.

Fialuridine (FIAU) is a nucleoside analog with potent activity against hepatitis B virus in vitro and in vivo. In this report, the effect of FIAU on mitochondrial DNA (mtDNA) replication in vitro was investigated. CEM cells, a cell line derived from human T cells, were incubated for 6 days in up to 20 microM FIAU. Total cellular DNA was isolated, normalized for the number of cells, and slot hybridized to a probe specific for mtDNA sequences. Treatment of CEM cells with FIAU did not result in a dose-dependent decrease in the amount of mtDNA. In contrast, dideoxycytidine (ddC) inhibited mtDNA replication by 50% at a concentration of approximately 0.1 microM. After 6 days of incubation, both compounds displayed a 50% toxic dose at a concentration of approximately 2 microM in CEM cells and approximately 34 microM in human hepatoblastoma cells (HepG2). In further experiments, CEM cells were incubated for 15 days in up to 2.5 microM FIAU, and again, no inhibition of mtDNA was observed. Over a 6-day incubation, FIAU, at concentrations of up to 200 microM, also failed to inhibit mtDNA replication in either HepG2 or HepG2 cells which constitutively replicate duck hepatitis B virus. In contrast, ddC inhibited mtDNA replication in these cells with a 50% inhibitory concentration of approximately 0.2 microM over a 6-day incubation. Treatment of cells with either FIAU or ddC resulted in a dose-dependent increase in lactate levels in the cell medium, indicating that any effect of FIAU on mitochondrial function may not be related to inhibition of mtDNA replication on the basis of the in vitro data. Alternative explanations for mitochondrial toxicity are considered.

A human hepatoblastoma cell line was stably transfected with a head-to-tail dimer of the Hepatitis B virus (HBV), subtype adw, genome to generate a cell line which produces HBV. FIAU [1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil] inhibited viral replication in these cells with an IC50 of 0.90 microM, as determined by PCR analysis of extracellular Dane particle DNA, and displayed a 50% cytotoxic concentration (TC50) of 344.3 microM, as determined using the MTT assay. The selectivity index of FIAU (TC50/IC50) was 382.6. In cells incubated for 10 days with FIAU (100 microM) and then incubated with drug-free media with daily media changes for 7 days, viral DNA replication was markedly inhibited but resumed within 24 h after drug removal, demonstrating that the in vitro anti-HBV activity of FIAU is reversible. Both the antiviral activity and cytotoxicity of FIAU were reversed by the addition of equimolar to 10-fold excess molar concentrations of thymidine. The de-iodinated metabolite of FIAU, FAU, had only marginal anti-HBV activity at 100 microM, indicating that this metabolite does not contribute significantly to the activity of FIAU. The examination of intracellular viral DNA replicative intermediates revealed that FIAU was 2000-fold more active against duck HBV DNA replication in human hepatoma cells (IC50 = 0.075 microM) than against this same virus in chicken liver cells (IC50 = 156 microM). FIAU was anabolized to a 25-fold greater extent in human hepatoma cells than in chicken cells, indicating that the anti-HBV activity of this nucleoside analog is dependent, in part, on its phosphorylation by the host cell.

Hypericin was found to be active against a member of the hepatitis B virus family, duck hepatitis B virus (DHBV). After a single 1 h incubation with hypericin, cells stably-transfected with a clone of DHBV stopped producing infectious virus for several days, though virus-like particles continued to be released into the culture medium. Characterization of these virions revealed a buoyant density characteristic of infectious virus preparations and lower than that of virus cores, suggesting that the particles were enveloped. Western blot analysis suggested, however, that the viral preS protein in surface antigen particles and, by inference, in virions, was present in covalently cross-linked aggregates. Evidence of a similar level of aggregation of the core subunit of virion nucleocapsids was not found, nor was there evidence of a similar high level of aggregation of cell-associated core and preS proteins. Hypericin was only slightly virucidal against DHBV and culture medium from treated cultures did not block initiation of infection when added to DHBV susceptible cultures prior to a challenge with infectious DHBV. Thus, the primary antiviral activity of hypericin against DHBV replication appears to be exerted at a late step in viral morphogenesis.