Acyclic Nucleotide Research Articles

Impact of Single Acyclic Phosphonate Nucleotide (ZNA) Modifications on DNA Duplex Stability.

An acyclic phosphonate-linked nucleic acid backbone (ZNA) demonstrated the capability to support duplex formation and propagate genetic information in vivo, unveiling its potential for evolution into a synthetic genetic system (XNA). To determine the structural impact of such modification, modified Dickerson Drew DNA dodecamers (DDDs) were prepared by solid phase synthesis, each containing either an (R) or (S) isomeric form of a cytosine ZNA nucleotide. While the DDD is known to adopt a stable duplex, both duplex and hairpin forms were simultaneously observed for both modified oligonucleotides by NMR spectroscopy over a broad temperature range (5-65 °C). Diffusion-ordered spectroscopy (DOSY) experiments allowed to separate duplex and hairpin signals based on the different diffusion constants of both conformational states. For the oligomer containing (R)-ZNA, only the duplex form occurred at 5 °C, while it was not possible to determine by NMR a single hairpin conformation at higher temperatures. In the case of the (S)-ZNA nucleoside modified oligomer, both hairpin and duplex forms were observable at 0 °C, while a single hairpin conformation was detected at 37 °C, suggesting a higher destabilizing effect on dsDNA.

Pharmacogenetics of tenofovir drug transporters in the context of HBV: Is there an impact?

BackgroundCurrent treatments for chronic hepatitis B management include orally administered nucleos(t)ide analogues, such as tenofovir (TDF), which is an acyclic adenine nucleotide analogue used both in HBV and human immune deficiency virus (HIV). The course of HBV infection is mainly dependent on viral factors, such as HBV genotypes, immunological features and host genetic variables, but a few data are available in the context of HBV, in particular for polymorphisms of genes encoding proteins involved in drug metabolism and elimination.Consequently, the aim of this study was to evaluate the potential impact of genetic variants on TDF plasma and urine concentrations in patients with HBV, considering the role of HBV genotypes. MethodsA retrospective cohort study at the Infectious Disease Unit of Amedeo di Savoia Hospital, Torino, Italy, was performed. Pharmacokinetic analyses were performed through liquidi chromatography, whereas pharmacogenetic analyses through real-time PCR. FindingsSixty - eight patients were analyzed: ABCC4 4976 C>T genetic variant showed an impact on urine TDF drug concentrations (p = 0.014). In addition, SLC22A6 453 AA was retained in the final regression multivariate model considering factors predicting plasma concentrations, while ABCC4 4976 TC/CC was the only predictor of urine concentrations in the univariate model. InterpretationIn conclusion, this is the first study showing a potential impact of genetic variants on TDF plasma and urine concentrations in the HBV context, but further studies in different and larger cohorts of patients are required.

Oral USC-093, a novel homoserinamide analogue of the tyrosinamide (S)-HPMPA prodrug USC-087 has decreased nephrotoxicity while maintaining antiviral efficacy against human adenovirus infection of Syrian hamsters

Adenovirus infections of immunocompromised humans are a significant source of morbidity and mortality. Presently, there is no drug specifically approved for the treatment of adenovirus infections by the FDA. The state-of-the-art treatment of such infections is the off-label use of cidofovir, an acyclic nucleotide phosphonate. While cidofovir inhibits adenovirus replication, it has dose-limiting kidney toxicity. There is an apparent need for a better compound to treat adenovirus infections. To this end, we have been developing acyclic nucleotide phosphonate prodrugs that utilize an amino acid scaffold equipped with a lipophilic modifier. Here, we compare the antiviral potential of two prodrugs of HPMPA that differ only in the amino acid-based promoiety: USC-087, based on an N-hexadecyl tyrosinamide, and USC-093, based on an N-hexadecyl serinamide. Oral administration of both compounds was very efficacious against disseminated HAdV-C6 infection in immunosuppressed Syrian hamsters, suppressing virus replication and mitigating pathology even when treatment was withheld until 4 days after challenge. We saw only marginal efficacy after respiratory infection of hamsters, which may reflect suboptimal distribution to the lung. Importantly, neither compound induced intestinal toxicity, which was observed as the major adverse effect in clinical trials of brincidofovir, a prodrug of cidofovir which also contains a C-16 modifier. Notably, we found that there was a significant difference in the nephrotoxicity of the two compounds: USC-087 caused significant kidney toxicity while USC-093 did not, at effective doses. These findings will be valuable guidepoints in the future evolution of this new class of potential prodrugs to treat adenovirus infections.

Nucleos(t)ide analogs for hepatitis B virus infection differentially regulate the growth factor signaling in hepatocytes.

Recent clinical studies have suggested that the risk of developing HCC might be lower in patients with chronic hepatitis B receiving tenofovir disoproxil fumarate than in patients receiving entecavir, although there is no difference in biochemical and virological remission between the 2 drugs. The effects of nucleoside analogs (NsAs; lamivudine and entecavir) or nucleotide analogs (NtAs; adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide) on cell growth and the expression of growth signaling molecules in hepatoma cell lines and PXB cells were investigated in vitro. The tumor inhibitory effects of NsAs or NtAs were evaluated using a mouse xenograft model, and protein phosphorylation profiles were investigated. The binding of NsAs or NtAs to the insulin receptor (INSR) was investigated by thermal shift assays. NtAs, but not NsAs, showed direct growth inhibitory effects on hepatoma cell lines in vitro and a mouse model in vivo. A phosphoprotein array revealed that INSR signaling was impaired and the levels of phosphorylated (p)-INSRβ and downstream molecules phosphorylated (p)-IRS1, p-AKT, p-Gab1, and p-SHP2 were substantially reduced by NtAs. In addition, p-epidermal growth factor receptor and p-AKT levels were substantially reduced by NtAs. Similar findings were also found in PXB cells and nontumor lesions of liver tissues from patients with chronic hepatitis B. Prodrug NtAs, but not their metabolites (adefovir, adefovir monophosphate, adefovir diphosphate, tenofovir, tenofovir monophosphate, and tenofovir diphosphate), had such effects. A thermal shift assay showed the binding of NtAs to INSRβ. NtAs (adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide), which are adenine derivative acyclic nucleotide analogs, potentially bind to the ATP-binding site of growth factor receptors and inhibit their autophosphorylation, which might reduce the risk of HCC in patients with chronic hepatitis B.

Membrane-permeable tenofovir-di- and monophosphate analogues

The development of new antiviral agents such as nucleoside analogues or acyclic nucleotide analogues (ANPs) and prodrugs thereof is an ongoing task. We report on the synthesis of three types of lipophilic triphosphate analogues of (R)-PMPA and dialkylated diphosphate analogues of (R)-PMPA. A highly selective release of the different nucleotide analogues ((R)-PMPA-DP, (R)-PMPA-MP, and (R)-PMPA) from these compounds was achieved. All dialkylated (R)-PMPA-prodrugs proved to be very stable in PBS as well as in CEM/0 cell extracts and human plasma. In primer extension assays, both the monoalkylated and the dialkylated (R)-PMPA-DP derivatives acted as (R)-PMPA-DP as a substrate for HIV-RT. In contrast, no incorporation events were observed using human polymerase γ. The dialkylated (R)-PMPA-compounds exhibited significant anti-HIV efficacy in HIV-1/2 infected cells (CEM/0 and CEM/TK−). Remarkably, the dialkylated (R)-PMPA-MP derivative 9a showed a 326-fold improved activity as compared to (R)-PMPA in HIV-2 infected CEM/TK− cells as well as a very high SI of 14,000. We are convinced that this study may significantly contribute to advancing antiviral agents developed based on nucleotide analogues in the future.

First transplantation of a pig heart from a multiple gene-modified donor, porcine cytomegalovirus/roseolovirus, and antiviral drugs.

First transplantation of a pig heart from a multiple gene-modified donor, porcine cytomegalovirus/roseolovirus, and antiviral drugs.

Imbalance of placental cyclic and acyclic nucleotides and modification of their relationship with the metabolism of regulatory gas-transmitters in preterm labor

Imbalance of placental cyclic and acyclic nucleotides and modification of their relationship with the metabolism of regulatory gas-transmitters in preterm labor

Renal Toxicity of Tenofovir: Narrative Review

Tenofovir is a reverse-transcriptase inhibitor based on acyclic nucleotide analogs. Tenofovir is a drug that is often used in treating HIV infection and has also been approved for treating infection by the hepatitis B virus. Despite the fact that its renal safety has been demonstrated in cell culture and clinical trials, clinical use and in vivo animal studies have shown its association with a low, but important, risk of kidney injury. Tenofovir accumulation in these mitochondria-rich cells is explained by proximal tubular cell secretion. Proximal tubular cell dysfunction is a symptom of Tenofovir nephrotoxicity, which might be the leading cause of acute renal injury or chronic diseases of the kidney. A review of articles is performed using keywords related to the topic in the databases of Google Scholar and PubMed, and 54 papers have been included, which were case studies, cross-sectional studies, and in vivo animal studies from 2004 up to 2021. The review aims at explaining the interaction of Tenofovir with kidney tubules, an association of genetic polymorphism, clinical features of Tenofovir-induced renal toxicity, potential mechanisms of Tenofovir-induced renal toxicity, its predisposing conditions and factors, and finally, some proposed strategies and agents to monitor and manage Tenofovir-induced nephrotoxicity.

Identification and Characterization of Besifovir-Resistant Hepatitis B Virus Isolated from a Chronic Hepatitis B Patient.

Hepatitis B virus (HBV) is known to cause severe liver diseases such as acute or chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis B (CHB) infection is a major health problem with nearly 300 million individuals infected worldwide. Currently, nucleos(t)ide analogs (NAs) and interferon alpha are clinically approved treatments for HBV infection. NAs are potent antiviral agents that bind to HBV polymerase and block viral reverse transcription and replication. Besifovir dipivoxil maleate (BSV) is a newly developed NA against HBV in the form of acyclic nucleotide phosphonate that is available for oral administration similar to adefovir and tenofovir. Until now, resistance to BSV treatment has not been reported. In this study, we found a CHB patient who showed viral breakthrough after long-term treatment with BSV. The isolated HBV DNA from patient’s serum were cloned into the replication-competent HBV 1.2 mer and the sequence of reverse transcriptase (RT) domain of HBV polymerase were analyzed. We also examined the drug susceptibility of generated clones in vitro. Several mutations were identified in HBV RT domain. A particular mutant harboring ten RT mutations showed resistance to BSV treatment in vitro. The ten mutations include rtV23I (I), rtH55R (R), rtY124H (H), rtD134E (E), rtN139K (K), rtL180M (M), rtM204V (V), rtQ267L (L), rtL269I (I) and rtL336M (M). To further identify the responsible mutations for BSV resistance, we performed in vitro drug susceptibility assay on several artificial clones. As a result, our study revealed that rtL180M (M) and rtM204V (V) mutations, already known as lamivudine-resistant mutations, confer resistance to BSV in the CHB patient.

Acyclic nucleoside phosphonates with adenine nucleobase inhibit Trypanosoma brucei adenine phosphoribosyltransferase in vitro

All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).

Method Development and Validation for Estimation of TinofovirDisproxil Fumarate in Pharmaceutical Preparation by UV-Spectroscopy and HPLC With Force Degradation Study

Tinofovir Disoproxil Fumarate (TDF) is an acyclic nucleotide diester analog of adenosine monophosphate from the antiviral category utilized in the AIDS and hepatitis B treatment. In the present study, two analytical methods, i.e. UV and HPLC were developed for the TDF’s estimation in pharmaceutical preparation. In the UV method, the mobile phase used was Methanol and water in 60:40 ratio for estimation of the drug at 260 nm and an assay of the (TDF) obtained was 99.53%. The method’s validation was carried out as per ICH Q2 R1 guidelines in which linearity was detected from 10-50 µg/ml range with a regression value of 0.999 Percent RSD value of accuracy, precision and robustness were below 2. In the HPLC method estimation of (TDF) was evaluated on Cosmosil C-18 (250mm×4.6ID, Particle size: 5 µ) column utilizing Methanol: Water (60:40), 0.9 ml/min of flow rate, detection wavelength was 260 nm and the time of retention observed was around 4.63 minutes with the assay value 99.07%. HPLC method was also validated according to ICH guidelines, where linearity was detected in the 10-50 μg/ml of range with the regression coefficient value 0.999. The % RSD of precision, accuracy, and robustness was below 2%. The study of forced degradation was also performed by the HPLC method utilizing methanol: water (60:40) at 260nm. From this study, it can be concluded that the developed methods for estimation of (TDF) drug in pharmaceutical preparation are simple, accurate, precise, and can be utilized in the routine analysis for quantification of the drug in a dosage form.

Discovery of new TLR7 agonists by a combination of statistical learning-based QSAR, virtual screening, and molecular dynamics

Search for new antiviral medications has surged in the past two years due to the COVID-19 crisis. Toll-like receptor 7 (TLR7) is among one of the most important TLR proteins of innate immunity that is responsible for broad antiviral response and immune system control. TLR7 agonists, as both vaccine adjuvants and immune response modulators, are among the top drug candidates for not only our contemporary viral pandemic but also other diseases. The agonists of TLR7 have been utilized as vaccine adjuvants and antiviral agents. In this study, we hybridized a statistical learning-based QSAR model with molecular docking and molecular dynamics simulation to extract new antiviral drugs by drug repurposing of the DrugBank database. First, we manually curated a dataset consisting of TLR7 agonists. The molecular descriptors of these compounds were extracted, and feature engineering was done to restrict the number of features to 45. We applied a statistically inspired modification of the partial least squares (SIMPLS) method to build our QSAR model. In the next stage, the DrugBank database was virtually screened structurally using molecular docking, and the top compounds for the guanosine binding site of TLR were identified. The result of molecular docking was again screened by the ligand-based approach of QSAR to eliminate compounds that do not display strong EC50 values by the previously trained model. We then subjected the final results to molecular dynamics simulation and compared our compounds with imiquimod (an FDA-approved TLR7 agonist) and compound 1 (the most active compound against TLR7 in vitro, EC50 = 0.2 nM). Our results evidently demonstrate that cephalosporins and nucleotide analogues (especially acyclic nucleotide analogues such as adefovir and cidofovir) are computationally potent agonists of TLR7. We finally reviewed some publications about cephalosporins that, just like pieces of a puzzle, completed our conclusion.

Force Degradation Study of Tenofovir Disoproxil Fumarate by UV-Spectrophotometric Method

Tenofovir disoproxil Fumarate (TDF), acyclic phosphonate nucleotide analogue, used as antiretroviral agents in the treatment of HIV-1 infection. A stability indicating UV -spectrophotometric method is simple, an accurate and economic, precise and reproducible method has been used for the estimation of Tenofovir disoproxil Fumarate in bulk and tablets dosage form in present work. The wavelength selected for the absorption correction method was 260 nm. The linearity range of 2-10μg/ml proved that it obeyed Beer’s Law and the correlation coefficient (r2) was found to be 0.999 at 260 nm. The drug was subjected to acid, alkali, peroxide, UV and Heat degradation. The force degradation studies of Tenofovir disoproxil Fumarate was done on Stress degradation by hydrolysis under alkaline condition by using 0.1N NAOH was found to be 10.6%. Stress degradation by hydrolysis under acidic condition by using 0.1N HCl and product degradation was found to be 10.95%. Oxidative degradation was done by using hydrogen peroxide and product degradation was found to be 12.22%. Neutral hydrolytic degradation was found to be 12.26%. Forced degradation studies of drug reveal good stability under the chosen experimental conditions.

Naturally‐Occurring Variants of Muscle‐Type Creatine Kinase Exhibit Altered Tenofovir Monophosphate Phosphorylation Activity

Tenofovir (TFV), prescribed as a disoproxil fumarate prodrug, is a component of the only FDA‐approved regimen for the prevention of HIV. TFV is an acyclic nucleotide analog reverse transcriptase inhibitor prodrug that mimics a nucleotide monophosphate in structure and must be phosphorylated twice by intracellular kinases to produce the pharmacologically active TFV‐diphosphate (DP) form. There have been clinical observations of variation in intracellular TFV‐DP levels, along with efficacy of TFV‐based HIV pre‐exposure prophylaxis (PrEP). We hypothesize that interindividual differences in the kinases that phosphorylate TFV may be a cause of these outcomes. We have elucidated the kinases responsible for TFV phosphorylation in tissues important to forming a barrier of protection against HIV infection, including muscle‐type creatine kinase (CKM), which is the primary kinase responsible for the phosphorylation of TFV‐monophosphate (MP) to the active TFV‐DP in colon tissue. In pursuit of understanding interindividual differences in these kinases, we sequenced the CKM gene in over 1000 patient samples and discovered several previously unreported single nucleotide variants, some of which lead to mutations in the CKM amino acid sequence. Using bioinformatic tools, we predicted that several of these mutants may markedly decrease enzyme function toward the endogenous substrate of the enzyme, ADP. We performed molecular docking simulations, using AutoDock, to model the binding of TFV‐MP to CKM and found that TFV‐MP binds to CKM much as does ADP, which TFV‐MP mimics in structure, suggesting that the bioinformatic predictions may also apply to changes in TFV‐MP phosphorylation activity of CKM. Docking results reveal a pocket of active site arginine residues at positions 130, 132, 236, 292, and 320 as likely important to charge stabilization of the phosphates of TFV‐MP. Notably, our sequencing revealed genetic variants producing R130H and R132C mutations that are predicted to decrease enzyme activity. In order to determine the effects of the naturally‐occurring point mutations on CKM activity toward TFV‐MP, wild‐type human CKM and 15 mutants from the genetic variation analysis were recombinantly expressed and purified. Then, in vitro assays were performed by incubating the proteins with TFV‐MP and measuring TFV‐DP formation via ultra‐high performance liquid chromatography tandem mass spectrometry. These results reveal 11 mutations in CKM that significantly decrease TFV‐MP phosphorylation. Three active site mutations, R130H, R132C, and N286I, along with W211R resulted in a decrease in TFV‐DP formation to only 3% of that of wild‐type CKM. Additionally, T35I, R43Q, I92M, H97Y, F169L, and Y173C mutations decreased activity to 14–25% of that of wild‐type. This evidence suggests that CKM genetics may play a role in interindividual variability in TFV activation and potentially the efficacy of TFV in PrEP settings.Support or Funding InformationThis research is funded by NIH R01 AI128781

Nucleoside Triphosphate Diphosphohydrolases Dephosphorylate the Pharmacologically Active Metabolite of Tenofovir

Tenofovir (TFV; prescribed as the tenofovir disoproxil fumarate prodrug), an acyclic nucleotide analog, inhibits human immunodeficiency virus (HIV) replication and is currently used for both HIV treatment and prevention (as pre‐exposure prophylaxis). The pharmacologically active form of TFV is TFV‐diphosphate (TFV‐DP). Previously, we reported the heterogeneous distribution of TFV and TFV‐DP in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV‐containing enema. Although the observed heterogeneity in TFV‐DP distribution in colorectal tissue is multifactorial, it could be envisioned that dephosphorylation plays a role in regulating TFV‐DP levels since this molecule is structurally similar to ATP, which is a substrate for nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases). Therefore, in order to gain increased understanding of TFV‐DP disposition, the ability of recombinant human NTPDase 1, 3, 4, 5, and 8 to dephosphorylate TFV‐DP was investigated by incubating these enzymes with TFV‐DP, individually. TFV‐DP levels were measured using an ultra‐high‐performance liquid chromatography tandem mass spectrometry approach. From these incubations, TFV‐DP substrate depletion, a measure of dephosphorylation, was observed in the presence of NTPDase 1 or 3 enzymes. The activities of NTPDase 1 and 3 towards TFV‐DP were further confirmed using a malachite green phosphate assay that measures the release of inorganic phosphate. Further, using immunoblotting, we tested whether NTPDase 1 and 3 are expressed in colorectal tissue. These studies revealed the presence of both proteins in colorectal tissue lysate. In order to gain an understanding of the spatial distribution patterns of NTPDase 1 and 3 in colorectal tissue, Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) was employed. As a first step, we developed a MALDI MSI method to directly detect and identify peptides of NTPDase 1 and 3 following trypsin digestion. Trypsin digestions on NTPDases were carried out using a concentration of 0.05 μg/μl, and α‐cyano‐4‐hydroxycinnamic acid was used as the MALDI matrix. Following trypsin digestions and MALDI MS analysis, peptides corresponding to NTPDase 1 and 3 were detected and identified. From these, NTPDase 1 exhibited peptides, SLSNYPFDFQGAR, SQHQETPVYLGATAGMR, and IITGQEEGAYGWITINYLLGK whereas NTPDase 3 yielded following peptides: GSGISSYGNNPQDVPR, YGIVLDAGSSR, FTEETWPQIHFEK, and MFTVLTR. Thus, in this study, we established a MALDI MS methodology for detection and identification of peptides corresponding to NTPDase 1 and 3. Furthermore, the above developed method can be used to generate spatial distribution profiles (including cell‐type and region‐specific localization patterns) of NTPDase 1 and 3 in colorectal and other tissues of interest that are susceptible to HIV infection. Data from in vitro incubations indicate that NTPDase 1 and 3 may contribute to the regulation of TFV‐DP levels.Support or Funding InformationThis work was funded by NIH grants U19AI113127, UM1 AI068613 and R01AI128781.

Design, synthesis and antiviral evaluation of novel acyclic phosphonate nucleotide analogs with triazolo[4,5-b]pyridine, imidazo[4,5-b]pyridine and imidazo[4,5-b]pyridin-2(3H)-one systems

A new series of phosphonylated triazolo[4,5-b]pyridine (1-deaza-8-azapurine), imidazo[4,5-b]pyridine (1-deazapurine) and imidazo[4,5-b]pyridin-2(3H)-one (1-deazapurin-8-one) were synthesized from 2-chloro-3-nitropyridine and selected diethyl ɷ-aminoalkylphosphonates followed by reduction of the nitro group and cyclization. In the final step O,O-diethylphosphonates were transformed into the corresponding phosphonic acids. All synthesized compounds were evaluated in vitro for inhibitory activity against a broad variety of DNA and RNA viruses and their cytotoxic potencies were also established. Compound 12f showed marginal activity against cytomegalovirus Davis strain (EC50 = 76.47 μM) in human embryonic lung (HEL) cells while compounds 10g (EC50 = 52.53 μM) and 12l (EC50 = 61.70 μM) were minimally active against the varicella-zoster virus Oka strain in HEL cells. Compounds under investigation were not cytotoxic at the maximum concentration evaluated (100 µM).

Identification of adenine-N9-(methoxy)ethyl-β-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes.

Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate)dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the Pα,β-bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1's catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-β-bisphosphonate, 10, was identified as an NPP1 inhibitor (Ki 16.3μM vs. the artificial substrate p-nitrophenyl thymidine-5'-monophosphate (p-Nph-5'-TMP), and 9.60μM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (Ki 12.6μM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100μM.

Interaction of Nucleotides with a Trinuclear Terbium(III)-Dizinc(II) Complex: Efficient Sensitization of Terbium Luminescence by Guanosine Monophosphate and Application to Real-Time Monitoring of Phosphodiesterase Activity.

An in-depth study of the interaction of a trinuclear terbium(III)-dizinc(II) complex with an array of nucleotides differing in the type of nucleobase and number of phosphate groups, as well as cyclic versus acyclic variants, is presented. The study examined the nature of the interaction and the efficiency at which guanine was able to sensitize terbium(III) luminescence. Competitive binding and titration studies were performed to help establish the nature/mode of the interactions. These established that (1) interaction occurs by the coordination of phosphate groups to zinc(II) (in addition to uridine in the case of uridine monophosphate), (2) acyclic nucleotides bind more strongly than cyclic counterparts because of their higher negative charge, (3) guanine-containing nucleotides are able to sensitize terbium(III) luminescence with the efficiency of sensitization following the order guanosine monophosphate (GMP) > guanosine diphosphate > guanosine triphosphate because of the mode of binding, and (4) nucleoside monophosphates bind to a single zinc(II) ion, whereas di- and triphosphates appear to bind in a bridging mode between two host molecules. Furthermore, it has been shown that guanine is a sensitizer of terbium(III) luminescence. On the basis of the ability of GMP to effectively sensitize terbium(III)-based luminescence while cyclic GMP (cGMP) does not, the complex has been utilized to monitor the catalytic conversion of cGMP to GMP by a phosphodiesterase enzyme in real time using time-gated luminescence on a benchtop fluorimeter. The complex has the potential to find broad application in monitoring the activity of enzymes that process nucleotides (co)substrates, including high-throughput drug-screening programs.

Acyclic Nucleoside Phosphonates Bearing (R)‐ or (S)‐9‐[3‐Hydroxy‐2‐(phosphonoethoxy)propyl] (HPEP) Moiety as Monomers for the Synthesis of Modified Oligonucleotides

Suitably protected (R)‐ and (S)‐HPEP {9‐[3‐hydroxy‐2‐(phosphonoethoxy)propyl]} derivatives containing adenine and thymine nucleobases were prepared as monomers for the introduction of acyclic nucleotide units into oligonucleotides. The solid phase synthesis of a series of ribo and 2′‐deoxyribo nonamers containing (R)‐HPEP and (S)‐HPEP units was successfully performed in both 5′ → 3′ and 3′ → 5′ directions. The measurement of thermal characteristics of the complexes of modified nonamers with complementary DNA and RNA strands revealed a general destabilizing effect of the introduced acyclic units on the thermal stability, but in several cases [modif.RNA*RNA and modif.DNA*RNA with (S)‐HPEPA units], the duplexes were characterized in terms of Tm values.

Intramolecular π-stacks in mixed-ligand copper(II) complexes formed by heteroaromatic amines and antivirally active acyclic nucleotide analogs carrying a hydroxy-2-(phosphonomethoxy)propyl residue‡

Acyclic nucleoside phosphonates (ANPs) are of medical relevance and deserve detailed chemical characterization. We focus here on 1-[2-(phosphonomethoxy)ethyl]cytosine (PMEC), (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC), 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), and (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) and include for comparison the nucleobase-free (phosphonomethoxy)ethane (PME) and (R)-hydroxy-2-(phosphonomethoxy)propane (HPMP). The acidity constants of H3(ANP)+ and H2(NP) (NP2– = nucleoside phosph(on)ate derivative) are needed to understand the properties of the ternary neutral Cu(Arm)(ANP/NP) [Arm = 2,2′-bipyridine (Bpy) or 1,10-phenanthroline (Phen)] and the monoprotonated Cu(Arm)(H;ANP)+ complexes. The Cu(Arm)(ANP) species are considerably more stable than the corresponding Cu(Arm)(R-PO3), where R- represents a phosph(on)ate ligand with a non-coordinating group R. The observed stability enhancements are due to intramolecular stack formation (st) between the aromatic rings of Arm and the nucleobase residues and also to the formation of five-membered chelates involving the ether oxygen of the –CH(R)–O–CH2– residue (cl/O) (R = H or CH2–OH). In intramolecular equilibria, three structurally different Cu(Arm)(ANP) isomers occur; for example, of Cu(Phen)(HPMPA) about 5% exist as an open (op) Cu(Phen)(HPMPA)op isomer, 17% as Cu(Phen)(HPMPA)cl/O, and 78% as Cu(Phen)(HPMPA)st. In Cu(Arm)(ANP) the stacking tendency decreases in the order PMEA2– > HPMPA2– > PMEC2– > HPMPC2–. In monoprotonated Cu(Arm)(H;ANP)+ both H+ and Cu(Arm)2+ are at undergoing similar intramolecular equilibria as indicated above.