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Chemical & Pharmaceutical Bulletin Dec 2022Quantitative H-NMR (H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for...
Quantitative H-NMR (H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for quantitation because of their overlap and complexity. Therefore, we focused on the P nucleus because of the simplicity of its signals and previously reported P-qNMR in DO. Here we report P-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-bis(trimethylsilyl)benzene-d (1,4-BTMSB-d) were used as reference standards for P-qNMR and H-qNMR, respectively, in methanol-d. The purity of SOF determined by P-qNMR was 100.63 ± 0.95%, whereas that determined by H-qNMR was 99.07 ± 0.50%. The average half bandwidths of the P signal of PAA and SOF were 3.38 ± 2.39 and 2.22 ± 0.19 Hz, respectively, suggesting that the T relaxation time of the PAA signal was shorter than that of SOF and varied among test laboratories. This difference most likely arose from the instability in the chemical shift due to the deuterium exchange of the acidic protons of PAA, which decreased the integrated intensity of the PAA signal. Next, an aprotic solvent, dimethyl sulfoxide-d (DMSO-d), was used as the dissolving solvent with PAA and sodium 4,4-dimethyl-4-silapentanesulfonate-d (DSS-d) as reference standards for P-qNMR and H-qNMR, respectively. SOF purities determined by P-qNMR and H-qNMR were 99.10 ± 0.30 and 99.44 ± 0.29%, respectively. SOF purities determined by P-qNMR agreed with the established H-qNMR values, suggesting that an aprotic solvent is preferable for P-qNMR because it is unnecessary to consider the effect of deuterium exchange.
Topics: Deuterium; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Reference Standards; Sofosbuvir; Solvents
PubMed: 36223954
DOI: 10.1248/cpb.c22-00639 -
Molecules (Basel, Switzerland) Sep 2022The chemical reactivity of 3-[()-3-(dimethylamino)-2-propenoyl]-4-hydroxy-1-methy-2(1)-quinolinone () towards some phosphorus reagents was studied. The enaminone was...
The chemical reactivity of 3-[()-3-(dimethylamino)-2-propenoyl]-4-hydroxy-1-methy-2(1)-quinolinone () towards some phosphorus reagents was studied. The enaminone was cyclized into pyranoquinolinylphosphonate via treatment with diethyl phosphite in basic medium. However, its reaction with triethoxy phosphonoacetate gave the substituted oxopyranylphosphonate . Using the same reaction conditions, both thioxopyridinylphosphonate and oxopyranylphosphonate were produced via a reaction of enaminone with both diethyl 2-amino-2-thioxoethylphosphonate and diethyl vinylphosphonate, respectively, in low yields. In addition, the two novel oxopyridinylphosphonates and were obtained by treatment of enaminone with a diethyl cyanomethylphosphonate reagent. Two oaxathiaphosphininyl derivatives, and were obtained by treatment of the enaminone with -diethyl dithiophosphoric acid under different reaction conditions. Diazaphosphininyl and oxazaphosphininyl derivatives were obtained in excellent yields using a -phenylphosphonic diamide reagent under different reaction conditions. The treatment of the enaminone with phosphorus pentasulfide produced the non-phosphorylated product thioxothiopyranoquinolinone . Finally, the enaminone was turned into oxathiaphosphininyl using Lawesson's reagent. The possible reaction mechanisms of the formation of these products were discussed. The structures of newly isolated products were established by elemental analysis and spectral tools. The compounds were evaluated for their antioxidant activities.
Topics: Antioxidants; Diamide; Indicators and Reagents; Organophosphonates; Phosphites; Phosphonoacetic Acid; Phosphorus; Quinolones
PubMed: 36144697
DOI: 10.3390/molecules27185960 -
Journal of Medical Virology Dec 2022HSV-2 antiviral resistance mainly occurs in immunocompromised patients and especially in HIV-positive individuals receiving long-term antiviral treatment. Those...
HSV-2 antiviral resistance mainly occurs in immunocompromised patients and especially in HIV-positive individuals receiving long-term antiviral treatment. Those situations can be challenging as few alternatives are available for HSV infection management. To describe clinical and virological significance of two novel potential HSV-2 resistance mutations after treating an obese patient with a pseudotumoral genital HSV-related lesion. Consecutive different antiviral treatments were used: valacyclovir (VACV) then foscarnet (FOS) then topical cidofovir (CDV) and finally imiquimod. Under VACV, genotypic resistance testing revealed a novel mutation within viral thymidine kinase (TK, gene UL23) not previously reported but probably accounting for antiviral resistance: W89G, similar to W88R mutation reported in HSV-1 TK, known to be associated with ACV resistance for HSV-1. Under FOS, while initial mutations were still present, a second genotypic resistance testing performed on persisting lesions showed a novel mutation within viral DNA polymerase (DNA pol, gene UL30): C625R. All three antivirals used in this case are small molecules and pharmacokinetics of VACV, FOS, and CDV have not been evaluated in animals and there are very few studies in human. As small molecules are poorly bound to proteins and distribution volume is increased in obese patients, there is risk of underdosage. This mechanism is suspected to be involved in emergence of resistance mutation and further data is needed to adapt, closely to patient profile, antiviral dosage. This report describes a chronic HSV-2 genital lesion, with resistance to current antivirals and novel mutations within viral TK and DNA pol which may confer antiviral resistance.
Topics: Acyclovir; Antiviral Agents; Cidofovir; DNA-Directed DNA Polymerase; Drug Resistance, Viral; Foscarnet; Genitalia; Herpes Simplex; Herpesvirus 2, Human; Humans; Imiquimod; Mutation; Obesity; Thymidine Kinase; Valacyclovir
PubMed: 35973907
DOI: 10.1002/jmv.28070 -
Antimicrobial Agents and Chemotherapy Sep 2022Maribavir was approved by the U.S. Food and Drug Administration in November 2021 for the treatment of adult and pediatric patients with post-transplant cytomegalovirus... (Review)
Review
Maribavir was approved by the U.S. Food and Drug Administration in November 2021 for the treatment of adult and pediatric patients with post-transplant cytomegalovirus (CMV) infection/disease that is refractory to treatment (with or without genotypic resistance) with ganciclovir, valganciclovir, cidofovir, or foscarnet. Maribavir is an oral benzimidazole riboside with potent and selective multimodal anti-CMV activity. It utilizes a novel mechanism of action which confers activity against CMV strains that are resistant to traditional anti-CMV agents, and also offers a more favorable safety profile relative to the dose-limiting side effects of previously available therapies. Maribavir was initially studied as an agent for CMV prophylaxis in solid organ and hematopoietic stem cell recipients, but initial phase III trials failed to meet clinical efficacy endpoints. It has been more recently studied as a therapeutic agent at higher doses for refractory-resistant (R-R) CMV infections with favorable outcomes. After an overview of maribavir's chemistry and clinical pharmacology, this review will summarize clinical efficacy, safety, tolerability, and resistance data associated with maribavir therapy.
Topics: Adult; Anti-Infective Agents; Antiviral Agents; Benzimidazoles; Child; Cidofovir; Cytomegalovirus Infections; Dichlororibofuranosylbenzimidazole; Drug Resistance, Viral; Foscarnet; Ganciclovir; Humans; Valganciclovir
PubMed: 35916518
DOI: 10.1128/aac.02405-21 -
Journal of Nanobiotechnology Jul 2022Cytomegalovirus (CMV) pneumonia is a major cause of morbidity and mortality in immunodeficiency individuals, including transplant recipients and Acquired Immune...
BACKGROUND
Cytomegalovirus (CMV) pneumonia is a major cause of morbidity and mortality in immunodeficiency individuals, including transplant recipients and Acquired Immune Deficiency Syndrome patients. Antiviral drugs ganciclovir (GCV) and phosphonoformate (PFA) are first-line agents for pneumonia caused by herpesvirus infection. However, the therapy suffers from various limitations such as low efficiency, drug resistance, toxicity, and lack of specificity.
METHODS
The antiviral drugs GCV and PFA were loaded into the pH-responsive nanoparticles fabricated by poly(lactic-co-glycolic acid) (PLGA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and further coated with cell membranes derived from bone marrow mesenchymal stem cells to form artificial stem cells, namely MPDGP. We evaluated the viral suppression effects of MPDGP in vitro and in vivo.
RESULTS
MPDGP showed significant inflammation tropism and efficient suppression of viral replication and virus infection-associated inflammation in the CMV-induced pneumonia model. The synergistic effects of the combination of viral DNA elongation inhibitor GCV and viral DNA polymerase inhibitor PFA on suppressing the inflammation efficiently.
CONCLUSION
The present study develops a novel therapeutic intervention using artificial stem cells to deliver antiviral drugs at inflammatory sites, which shows great potential for the targeted treatment of pneumonia. To our best knowledge, we are the first to fabricate this kind of artificial stem cell to deliver antiviral drugs for pneumonia treatment.
Topics: Antiviral Agents; Cytomegalovirus; Cytomegalovirus Infections; Fatty Acids, Monounsaturated; Foscarnet; Ganciclovir; Humans; Inflammation; Nanoparticle Drug Delivery System; Pneumonia; Polylactic Acid-Polyglycolic Acid Copolymer; Quaternary Ammonium Compounds; Stem Cells
PubMed: 35842662
DOI: 10.1186/s12951-022-01547-x -
Journal of Virology Jul 2022Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the family. We showed earlier that infection of porcine epithelial cells with PRV triggers...
Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the family. We showed earlier that infection of porcine epithelial cells with PRV triggers activation of the nuclear factor κB (NF-κB) pathway, a pivotal signaling axis in the early immune response. However, PRV-induced NF-κB activation does not lead to NF-κB-dependent gene expression. Here, using electrophoretic mobility shift assays (EMSAs), we show that PRV does not disrupt the ability of NF-κB to interact with its κB target sites. Assessing basal cellular transcriptional activity in PRV-infected cells by quantitation of prespliced transcripts of constitutively expressed genes uncovered a broad suppression of cellular transcription by PRV, which also affects the inducible expression of NF-κB target genes. Host cell transcription inhibition was rescued when viral genome replication was blocked using phosphonoacetic acid (PAA). Remarkably, we found that host gene expression shutoff in PRV-infected cells correlated with a substantial retention of the NF-κB subunit p65, the TATA box binding protein, and RNA polymerase II-essential factors required for (NF-κB-dependent) gene transcription-in expanding PRV replication centers in the nucleus and thereby away from the host chromatin. This study reveals a potent mechanism used by the alphaherpesvirus PRV to steer the protein production capacity of infected cells to viral proteins by preventing expression of host genes, including inducible genes involved in mounting antiviral responses. Herpesviruses are highly successful pathogens that cause lifelong persistent infections of their host. Modulation of the intracellular environment of infected cells is imperative for the success of virus infections. We reported earlier that a DNA damage response in epithelial cells infected with the alphaherpesvirus pseudorabies virus (PRV) results in activation of the hallmark proinflammatory NF-κB signaling axis but, remarkably, that this activation does not lead to NF-κB-induced (proinflammatory) gene expression. Here, we report that PRV-mediated inhibition of host gene expression stretches beyond NF-κB-dependent gene expression and in fact reflects a broad inhibition of host gene transcription, which correlates with a substantial recruitment of essential host transcription factors in viral replication compartments in the nucleus, away from the host chromatin. These data uncover a potent alphaherpesvirus mechanism to interfere with production of host proteins, including proteins involved in antiviral responses.
Topics: Animals; Herpesvirus 1, Suid; Host Microbial Interactions; NF-kappa B; Pseudorabies; Swine; Swine Diseases; Transcription, Genetic
PubMed: 35730976
DOI: 10.1128/jvi.00714-22 -
Biochemistry Dec 2023CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the...
CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'--methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'--methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.
Topics: Humans; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Phosphonoacetic Acid; Gene Editing; RNA, Messenger
PubMed: 35436085
DOI: 10.1021/acs.biochem.1c00768 -
Kidney360 Jul 2021
Topics: Allografts; Foscarnet; Kidney; Transplantation, Homologous
PubMed: 35368358
DOI: 10.34067/KID.0001062021 -
ACS Biomaterials Science & Engineering Apr 2022[ZrO][(FCN)(OH)] and Gd[FCN] inorganic-organic hybrid nanoparticles (IOH-NPs) are novel saline antiviral nanocarriers with foscarnet (FCN) as a drug anion. FCN as a...
[ZrO][(FCN)(OH)] and Gd[FCN] inorganic-organic hybrid nanoparticles (IOH-NPs) are novel saline antiviral nanocarriers with foscarnet (FCN) as a drug anion. FCN as a pyrophosphate analogue serves as a prototype of a viral DNA polymerase inhibitor. FCN is used for the treatment of herpesvirus infections, including the drug-resistant cytomegalovirus (CMV) and herpes simplex viruses, HSV-1 and HSV-2. The novel [ZrO][(FCN)(OH)] and Gd[FCN] IOH-NPs are characterized by aqueous synthesis, small size (20-30 nm), low material complexity, high biocompatibility, and high drug load (up to 44 wt % FCN per nanoparticle). The antiviral activity of the FCN-type IOH-NPs is probed for the human cytomegalovirus (HCMV). Moreover, the uptake of FCN-type IOH-NPs into vesicles, cytoplasm, and nuclei of nonphagocytic lung epithelial cells is evaluated. As a result, a promising antiviral activity of the FCN-type IOH-NPs that significantly outperforms freely dissolved FCN at the level of clinical formulations is observed, encouraging a future use of FCN-type IOH-NPs for the delivery of antivirals against respiratory viruses.
Topics: Antiviral Agents; Cytomegalovirus; Foscarnet; Herpesvirus 1, Human; Humans; Nanoparticles
PubMed: 35344659
DOI: 10.1021/acsbiomaterials.2c00074 -
Biomedicines Mar 2022Despite the eradication of smallpox four decades ago, poxviruses continue to be a threat to humans and animals. The arsenal of anti-poxvirus agents is very limited and...
Despite the eradication of smallpox four decades ago, poxviruses continue to be a threat to humans and animals. The arsenal of anti-poxvirus agents is very limited and understanding mechanisms of resistance to agents targeting viral DNA polymerases is fundamental for the development of antiviral therapies. We describe here the phenotypic and genotypic characterization of poxvirus DNA polymerase mutants isolated under selective pressure with different acyclic nucleoside phosphonates, including HPMPC (cidofovir), cHPMPC, HPMPA, cHPMPA, HPMPDAP, HPMPO-DAPy, and PMEO-DAPy, and the pyrophosphate analogue phosphonoacetic acid. Vaccinia virus (VACV) and cowpox virus drug-resistant viral clones emerging under drug pressure were characterized phenotypically (drug-susceptibility profile) and genotypically (DNA polymerase sequencing). Different amino acid changes in the polymerase domain and in the 3'-5' exonuclease domain were linked to drug resistance. Changes in the 3'-5' domain emerged earlier than in the polymerase domain when viruses acquired a combination of mutations. Our study highlights the importance of poxvirus DNA polymerase residues 314, 613, 684, 688, and 851, previously linked to drug resistance, and identified several novel mutations in the 3'-5' exonuclease domain (M313I, F354L, D480Y) and in the DNA polymerase domain (A632T, T831I, E856K, L924F) associated with different drug-susceptibility profiles. Furthermore, a combination of mutations resulted in complex patterns of cross-resistance. Modeling of the VACV DNA polymerase bearing the newly described mutations was performed to understand the effects of these mutations on the structure of the viral enzyme. We demonstrated the emergence of drug-resistant DNA polymerase mutations in complex patterns to be considered in case such mutations should eventually arise in the clinic.
PubMed: 35327382
DOI: 10.3390/biomedicines10030580