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The unprecedented magnitude of the 2013-2016 Ebola virus (EBOV) epidemic in West Africa resulted in over 11,000 deaths and spurred an international public health emergency. A second outbreak in 2018-2020 in DRC resulted in an additional >3400 cases and nearly 2300 deaths (WHO Disease Outbreak News: Update 26 June, 2020). These large outbreaks across geographically diverse regions highlight the need for the development of effective oral therapeutic agents that can be easily distributed for self-administration to populations with active disease or at risk of infection. Herein, we report the in vivo efficacy ofN4-hydroxycytidine (EIDD-1931), a broadly active ribonucleoside analog and the active metabolite of the prodrug EIDD-2801 (molnupiravir), in murine models of lethal EBOV infection. Twice daily oral dosing with EIDD-1931 at 200 mg/kg for 7 days, initiated either with a prophylactic dose 2 hours before infection, or as therapeutic treatment starting 6 hours post-infection, resulted in 92-100% survival of mice challenged with lethal doses of EBOV, reduced clinical signs of Ebola virus disease (EVD), reduced serum virus titers, and facilitated weight loss recovery. These results support further investigation of molnupiravir as a potential therapeutic or prophylactic treatment for EVD.

ATI-2173 is an active site polymerase inhibitor nucleotide in development as part of a potentially curative regimen for chronic hepatitis B virus (HBV) infection. This study evaluated the safety, tolerability, pharmacokinetics (PK) and antiviral activity of ATI-2173. This was a phase 1b, randomized, double-blind, placebo-controlled trial in treatment-naive adults with chronic HBV infection conducted in the Republic of Moldova and Ukraine (ClinicalTrials.gov: NCT04248426). Patients positive for hepatitis B surface antigen were randomized 6:2 to receive once-daily oral doses of ATI-2173 10, 25, or 50 mg (n = 6 per dose) or placebo (n = 7) for 28 days, with off-treatment monitoring for 24 weeks. Endpoints included PK parameters of ATI-2173 and its metabolite clevudine, maximum reduction from baseline in HBV DNA, and safety and tolerability. Treatment-emergent adverse events occurred in eight patients (47%) receiving ATI-2173 and five (71%) receiving placebo; headache was the most common (n = 4). ATI-2173 PK was generally dose proportional. Systemic clevudine exposure with ATI-2173 dosing was substantially reduced compared with historical values observed with clevudine administration. On Day 28, mean changes from baseline in HBV DNA were -2.72 to -2.78 log

The unprecedented magnitude of the 2013-2016 Ebola virus (EBOV) epidemic in West Africa resulted in over 11 000 deaths and spurred an international public health emergency. A second outbreak in 2018-2020 in DRC resulted in an additional3400 cases and nearly 2300 deaths (WHO, 2020). These large outbreaks across geographically diverse regions highlight the need for the development of effective oral therapeutic agents that can be easily distributed for self-administration to populations with active disease or at risk of infection. Herein, we report the in vivo efficacy of N

ATI-2173 is a novel liver-targeted molecule designed to deliver the 5′-monophosphate of clevudine for the treatment of chronic hepatitis B infection. Unlike other nucleos(t)ides, the active clevudine-5′-triphosphate is a noncompetitive, non-chain-terminating inhibitor of hepatitis B virus (HBV) polymerase that delivers prolonged reduction of viremia in both a woodchuck HBV model and in humans for up to 6 months after cessation of treatment. However, long-term clevudine treatment was found to exhibit reversible skeletal myopathy in a small subset of patients and was subsequently discontinued from development., ATI-2173 is a novel liver-targeted molecule designed to deliver the 5′-monophosphate of clevudine for the treatment of chronic hepatitis B infection. Unlike other nucleos(t)ides, the active clevudine-5′-triphosphate is a noncompetitive, non-chain-terminating inhibitor of hepatitis B virus (HBV) polymerase that delivers prolonged reduction of viremia in both a woodchuck HBV model and in humans for up to 6 months after cessation of treatment. However, long-term clevudine treatment was found to exhibit reversible skeletal myopathy in a small subset of patients and was subsequently discontinued from development. ATI-2173 was designed by modifying clevudine with a 5′-phosphoramidate to deliver the 5′-monophosphate to the liver. Bypassing the first phosphorylation step of clevudine, the 5′-monophosphate is converted to the active 5′-triphosphate in the liver. ATI-2173 is a selective inhibitor of HBV with an anti-HBV 50% effective concentration (EC50) of 1.31 nM in primary human hepatocytes, with minimal to no toxicity in hepatocytes, skeletal muscle, liver, kidney, bone marrow, and cardiomyocytes. ATI-2173 activity was decreased by viral polymerase mutations associated with entecavir, lamivudine, and adefovir resistance, but not capsid inhibitor resistance mutations. A single oral dose of ATI-2173 demonstrated 82% hepatic extraction, no food effect, and greatly reduced peripheral exposure of clevudine compared with equimolar oral dosing of clevudine. Despite reduced plasma clevudine exposure, liver concentrations of the 5′-triphosphate were equivalent following ATI-2173 versus clevudine administration. By selectively delivering the 5′-monophosphate to the liver, while retaining the unique anti-HBV activity of the 5′-triphosphate, ATI-2173 may provide an improved pharmacokinetic profile for clinical use, reducing systemic exposure of clevudine and potentially eliminating skeletal myopathy.

There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5′-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5′-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination ( D ) values of ribonucleotide analog 5′-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5′-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound has a D value of >10 5 ; a potentially toxic compound has a D value of >10 4 but 5 ; and a toxic compound has a D value of 4 . This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.

Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn 2+ is required for enzymatic activity, while Mg 2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

Introduction. RG7128 (prodrug of PSI-6130) shows potent antiviral efficacy in patients infected with hepatitis C virus (HCV) genotypes 1, 2, or 3, with mean viral load decreases of 2.7 and 5 log 10 IU/mL, respectively, associated with 1500-mg doses twice daily after monotherapy for 2 weeks and with 1000-mg and 1500-mg doses twice daily after treatment in combination with the standard of care (SOC) for 4 weeks.Results. From 32 patients treated with RG7128 monotherapy for 2 weeks, marginal viral load rebound was observed in 3 HCV genotype 1-infected patients, whereas partial response was observed in 2 genotype 1-infected patients. From 85 patients receiving RG7128 in combination with SOC, 1 HCV genotype 1-infected patient experienced a viral rebound, and 2 genotype 3-infected patients experienced a transient rebound. Five genotype 1-infected patients had an HCV load of >1000 IU/mL at the end of 4-week treatment. No viral resistance was observed, per NS5B sequencing and phenotypic studies. PSI-6130 resistance substitution S282T needs to be present at levels of ⩾90% within a patient's quasispecies to confer low-level resistance. No evidence of S282T was found by population or clonal sequence analyses.Conclusions. The requirement for a predominant S282T mutant quasispecies, its low replication capacity, and the low-level resistance it confers probably contribute to the lack of RG7128 resistance observed in HCV-infected patients.

Reverset™ (2′,3′-didehydro-2′,3′-dideoxy-5-fluorocyti-dine, RVT) is a potent inhibitor of HIV-1 replication in cell culture, with a 90% effective concentration at or below 1 μM. In vitro, RVT retains its activity against isolates harbouring mutations in the reverse transcriptase (RT) gene that otherwise confer resistance to lamivudine and/or zidovudine. The pharmacokinetics and safety of single oral doses of RVT (10–200 mg) were evaluated in an initial Phase I clinical trial. The viral load changes were determined on 18 HIV-1-infected antiretroviral therapy-naive subjects that were randomized into three cohorts, each cohort consisting of three study periods. The subjects received up to two oral doses of active drug and one placebo dose with a 1-week washout period separating the three study periods. Quantification of viral RNA was performed on the pre-dose, 12, 24 and 48 h post-dose plasma samples. A single oral dose of RVT to antiretroviral-naive subjects significantly reduced plasma viral load by 0.45 ±0.10 log10 copies/ml ( P=0.0003). A mean drop of 0.37 ±0.12 log10 copies/ml ( P=0.001) was obtained at the lowest dose of 10 mg. Sequence analysis of the HIV-1 RT gene performed before and after RVT dosing detected no genotypic changes in this short-term study. The viral RT gene of one subject had at predose the following genotype: L41 + N103 + C181 + W210 + D215, indicating prior exposure to zidovudine and non-nucleoside analogues, and anticipating high-level resistance against these agents. A single 10 mg RVT dose resulted in a viral load drop of 0.61 ±0.05 log10 providing evidence that a viral strain with the indicated genotype is susceptible to RVT.

Objective: In this study we evaluated the possibility that plasma viral load elevations secondary to influenza vaccination in HIV-1-seropositive individuals with previously undetectable viral loads (< 200 copies/ml) could develop resistance-bearing mutations in the viral reverse transcriptase (RT) and protease regions. Methods: Thirty-four patients with undetectable viral burdens on highly active antiretroviral therapy (HAART) were evaluated for elevations in plasma viral load 2 and 4 weeks post-influenza vaccination. Plasma from patients whose viral load increased after vaccination was subject to genotypic resistance analysis by the line probe assay (LiPA) to determine whether primary resistance-bearing mutations developed during this period and at follow-up. Stored plasma was used to evaluate whether RT or protease mutations existed pre-vaccination. Results: Seven out of 34 patients were found to experience elevations in their viral load after influenza vaccination. Two of the patients revealed evidence of primary RT or protease mutations not demonstrated in earlier pre-vaccination samples. One patient failed therapy after vaccination, and one patient revealed post-vaccination viral load elevations that eventually led to the progressive development of primary zidovudine mutations. Conclusion: Evidence is presented that supports the contention that a small subset of patients who experience viral load elevations after influenza vaccination can develop mutational changes in the RT region of the viral genome either acutely or after a failure of the viral load to return to undetectable levels.

We hypothesize that elevation of nm23-H1 metastasis suppressor gene expression in micrometastatic tumor cells may reduce their subsequent colonization and invasion, and induce differentiation, with a clinical benefit. This report presents the first analysis of the nm23-H1 promoter to identify sites which can increase its transcription. Deletion mapping of a 2.1 kb nm23-H1 promoter fragment tethered to a reporter gene identified three regions involved in its differential expression levels among a panel of human breast carcinoma cell lines: a 195 bp NheI-XbaI fragment responsible for basal expression levels, a 248 bp AvrII-NheI fragment which contributed to the elevated nm23-H1 expression observed in the high expressing cell lines, and a 544 bp AvrII fragment containing an inhibitory element. Examination of the 248 bp AvrII-NheI fragment revealed the unexpected presence of three transcription factor binding sites (MAF/Ets, CTF/NF1 half site and ACAAAG enhancer) previously identified in the MMTV-LTR, and in WAP and milk gene promoters, proposed to mediate mammary-specific gene expression. Mutation of the three sites, individually or together, resulted in two-fold reductions in reporter gene expression. As controls, the same panel of mutations caused a different pattern of reporter gene expression in a non-mammary cell line, and mutation of another nearby site was without effect on nm23-H1. Our data identify a complex regulatory pattern for nm23-H1 transcription, and suggest that a mammary-specific cassette of transcription factors contribute to its elevated expression

Metastasis is suppressed more than 95% following microcell-mediated transfer of a single copy of neomycin-tagged human chromosome 6 (neo6) into the human melanoma cell lines C8161 and MelJuSo. Concomitant with metastasis suppression is upregulation of NME1 (Nm23-H1) mRNA and protein expression. The purposes of this study were to determine whether NME1 expression was responsible for metastasis suppression in neo6/melanoma hybrids, and whether genes on chromosome 6 regulate NME1. Using neo6/C8161 cells, transfection of CAT reporter constructs linked to the NME1 promoter failed to consistently induce CAT. Therefore, it does not appear that genes on chromosome 6 directly control transcription of NME1. Transfection and overexpression of NME1 in MelJuSo, under the control of the CMV promoter, resulted in 40-80% inhibition of lung metastasis following i.v. inoculation of 2´10 cells. Only one transfectant of C8161 subclone 9 (C8161cl.9) cells was suppressed for metastasis. Control transfections with pCMVneo or pSV2neo did not suppress metastasis in either cell line. Taken together, these data suggest that NME1 can reduce metastatic potential of some human melanoma cells; but, this inhibitory activity appears to be independent of the metastasis suppression following introduction of chromosome 6 into C8161 and MelJuSo human melanoma cell lines.

Reduced expression of nm23 has been associated with increased metastases and decreased survival in a variety of malignancies. In the present study, the expression of nm23 was examined by Northern and Western blot analyses in a series of cell lines derived from patients with metastatic renal cell carcinoma. Two of twelve (17%) informative cell lines derived from 9 patients had loss of heterozygosity at Nm23-H1. Twenty-two renal cancer cell lines derived from primary tumors, 5 cell lines derived from metastatic tumors and 4 short-term cultures of normal proximal renal tubular cells all expressed Nm23 mRNA in varying amounts. On average, the level of expression of Nm23 mRNA in short-term cultures of benign proximal renal tubular cells was found to be similar to the level seen in renal cancer cell lines. Twenty-eight cell lines derived from renal primary tumors and 8 cell lines derived from metastatic tumors expressed both the Nm23-H1 and Nm23-H2 proteins. High or low relative expression of nm23 at the mRNA or protein level did not correlate with survival. The absence of any anomalous pattern of expression of the nm23 genes and the lack of correlation of expression with survival suggests that nm23 does not play a central role in the progression of this tumor type.

Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]prothymosin alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]prothymosin alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine prothymosin alpha and human [32P]prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.

The role of sexual or intrafamilial transmission of hepatitis C is controversial. A phylogenetic analysis was performed on the non-structural region 5B of the hepatitis C virus (NS5B-HCV). High percentages of homology (mean of 98.3%) were shown between the couples. Twenty (83.3%) of the 24 men but only two of the women (8.3%) reported having had sexually transmitted diseases during their lives. The risk factors for HCV acquisition were blood transfusion (10 couples), use of illegal injected drugs (17), use of inhalants (15), acupuncture (5) and tattoos (5). The shared use of personal hygiene items included toothbrushes between six couples (25%), razor blades between 16 (66.7%), nail clippers between 21 (87.5%) and manicure pliers between 14 (58.3%). The high degree of similarity of the hepatitis C virus genome supports the hypothesis of hepatitis C virus transmission between these couples. The shared use of personal hygiene items suggests the possibility of intrafamilial transmission of infection.

Reverset (2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, RVT) is a potent inhibitor of HIV-1 replication in cell culture, with a 90% effective concentration at or below 1 microM. In vitro, RVT retains its activity against isolates harbouring mutations in the reverse transcriptase (RT) gene that otherwise confer resistance to lamivudine and/or zidovudine. The pharmacokinetics and safety of single oral doses of RVT (10-200 mg) were evaluated in an initial Phase I clinical trial. The viral load changes were determined on 18 HIV-1-infected antiretroviral therapy-naive subjects that were randomized into three cohorts, each cohort consisting of three study periods. The subjects received up to two oral doses of active drug and one placebo dose with a 1-week washout period separating the three study periods. Quantification of viral RNA was performed on the pre-dose, 12, 24 and 48 h post-dose plasma samples. A single oral dose of RVT to antiretroviral-naive subjects significantly reduced plasma viral load by 0.45 +/- 0.10 log10 copies/ml (P=0.0003). A mean drop of 0.37 +/- 0.12 log10 copies/ml (P=0.001) was obtained at the lowest dose of 10 mg. Sequence analysis of the HIV-1 RT gene performed before and after RVT dosing detected no genotypic changes in this short-term study. The viral RT gene of one subject had at predose the following genotype: L41 + N103 + C181 + W210 + D215, indicating prior exposure to zidovudine and non-nucleoside analogues, and anticipating high-level resistance against these agents. A single 10 mg RVT dose resulted in a viral load drop of 0.61 +/- 0.05 log10 providing evidence that a viral strain with the indicated genotype is susceptible to RVT.

We hypothesize that elevation of nm23-HI metastasis suppressor gene expression in micrometastatic tumor cells may reduce their subsequent colonization and invasion, and induce differentiation, with a clinical benefit. This report presents the first analysis of the nm23-HI promoter to identify sites which can increase its transcription. Deletion mapping of a 2.1 kb nm23-H1 promoter fragment tethered to a reporter gene identified three regions involved in its differential expression levels among a panel of human breast carcinoma cell lines: a 195 bp NheI-XbaI fragment responsible for basal expression levels, a 248 bp AvrII-Nhel fragment which contributed to the elevated nm23-H1 expression observed in the high expressing cell lines, and a 544 bp AvrII fragment containing an inhibitory element. Examination of the 248 bp AvrII-NheI fragment revealed the unexpected presence of three transcription factor binding sites (MAF/Ets, CTF/NF1 half site and ACAAAG enhancer) previously identified in the MMTV-LTR, and in WAP and milk gene promoters, proposed to mediate mammary-specific gene expression. Mutation of the three sites, individually or together, resulted in two-fold reductions in reporter gene expression. As controls, the same panel of mutations caused a different pattern of reporter gene expression in a non-mammary cell line, and mutation of another nearby site was without effect on nm23-HI. Our data identify a complex regulatory pattern for nm23-H1 transcription, and suggest that a mammary-specific cassette of transcription factors contribute to its elevated expression

Since the early 1970s, researchers have used nucleic acid detection techniques to identify either specific genes or organisms. These early nucleic acid detection methods were very time-consuming, cumbersome, isotopic, technically elaborate, and only moderately sensitive, and thus not suitable for routine clinical use (Southern, 1975). The first nonisotopic nucleic acid detection systems, developed in the late 1970s, were fairly specific but not as sensitive as their radioactive counterparts (Matthews and Kricka, 1988). To increase overall sensitivity, amplification methodologies have been developed and adapted to nonisotopic nucleic acid detection assays. The two most common amplification methods are target amplification (Mullis and Faloona, 1987) and signal amplification employing chemiluminescence (Pollard-Knight et al., 1990; Bronstein and Olesen, 1995).

The nm23 gene, a putative metastasis suppressor gene, was originally identified by its reduced expression in highly metastatic K-1735 murine melanoma cell lines, as compared to related, low metastatic melanoma cell lines. Transfection of nm23 cDNA has been reported to suppress malignant progression in Drosophila and mammalian cells. Highly conserved homologues of nm23 have been found in organisms ranging from the prokaryote Myxococcus xanthus to Drosophila, where the gene is involved in normal development and differentiation. The product of the nm23 gene exhibits a nucleoside diphosphate kinase activity, yet the nucleoside diphosphate kinase activity of Nm23 does not correlate with its apparent biological functions. We review recent cellular, genetic, biochemical and X-ray crystallographic data to formulate and evaluate hypotheses concerning the molecular mechanism of nm23 action.