Inosine Analogs Research Articles

Fluorescent inosine analogues: Synthesis, cytotoxicity activity and self-assembly nanoparticle for live cell image

The fluorescence is of importance for the investigation of pharmacokinetic of nanomedicines. The fluorescent DOI nanoparticles was obtained by self-assembly of amphiphilic 2′,5′-dioleoyl 8-(4-(p-ethoxyphenylethynyl) phenyl)-inosine 5 (DOI) which was synthesized by coupling two oleic acid chains to the sugar moiety of fluorescent inosine analogue synthesized by Suzuki cross-coupling reaction. IC50 value on A549 cells of DOI nanoparticles was 10.99 μg/mL which was much lower than that of free nucleoside 4e (31.16 μg/mL) owing to improving in vitro cell uptake, which was examined via autofluorescence of nucleosides 4e and DOI nanoparticle in live cells. This indicates that the bright fluorescent nucleosides can be used for live cell image and has the potential for investigating pharmacokinetic characteristics of nucleoside nanomedicines.

Nucleoside analogues for the treatment of animal trypanosomiasis

Animal trypanosomiasis (AT) is a parasitic disease with high socio-economic impact. Given the limited therapeutic options and problems of toxicity and drug resistance, this study assessed redirecting our previously identified antitrypanosomal nucleosides for the treatment of AT. Promising hits were identified with excellent in vitro activity across all important animal trypanosome species. Compound 7, an inosine analogue, and our previously described lead compound, 3′-deoxytubercidin (8), showed broad spectrum anti-AT activity, metabolic stability in the target host species and absence of toxicity, but with variable efficacy ranging from limited activity to full cure in mouse models of Trypanosoma congolense and T. vivax infection. Several compounds show promise against T. evansi (surra) and T. equiperdum (dourine). Given the preferred target product profile for a broad-spectrum compound against AT, this study emphasizes the need to include T. vivax in the screening cascade given its divergent susceptibility profile and provides a basis for lead optimization towards such broad spectrum anti-AT compound.

Inhibition of the hexamerization of SARS-CoV-2 endoribonuclease and modeling of RNA structures bound to the hexamer

Non-structural protein 15 (Nsp15) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) forms a homo hexamer and functions as an endoribonuclease. Here, we propose that Nsp15 activity may be inhibited by preventing its hexamerization through drug binding. We first explored the stable conformation of the Nsp15 monomer as the global free energy minimum conformation in the free energy landscape using a combination of parallel cascade selection molecular dynamics (PaCS-MD) and the Markov state model (MSM), and found that the Nsp15 monomer forms a more open conformation with larger druggable pockets on the surface. Targeting the pockets with high druggability scores, we conducted ligand docking and identified compounds that tightly bind to the Nsp15 monomer. The top poses with Nsp15 were subjected to binding free energy calculations by dissociation PaCS-MD and MSM (dPaCS-MD/MSM), indicating the stability of the complexes. One of the identified pockets, which is distinctively bound by inosine analogues, may be an alternative binding site to stabilize viral RNA binding and/or an alternative catalytic site. We constructed a stable RNA structure model bound to both UTP and alternative binding sites, providing a reasonable proposed model of the Nsp15/RNA complex.

Synthesis and evaluation of a collection of purine-like C-nucleosides as antikinetoplastid agents.

The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are the causative agents of neglected tropical diseases with a serious burden in several parts of the world. These parasites are incapable of synthesizing purines de novo, and therefore rely on ingenious purine salvage pathways to acquire and process purines from their host. Purine nucleoside analogs that may interfere with these pathways therefore constitute a privileged source of new antikinetoplastid agents. In this study, we synthetized a collection of C-nucleosides employing five different heterocyclic nucleobase surrogates. C-nucleosides are chemically and enzymatically stable and allow for extensive structural modification. Inspired by earlier 7-deazaadenosine nucleosides and known antileishmanial C-nucleosides, we introduced different modifications tailored towards antikinetoplastid activity. Both adenosine and inosine analogs were synthesized with the aim of discovering new antikinetoplastid hits and expanding knowledge of structure-activity relationships. Several promising hits with potent activity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum were discovered, and the nature of the nucleobase surrogate was found to have a profound influence on the selectivity profile of the compounds.

Adaptive evolution at mRNA editing sites in soft-bodied cephalopods.

BackgroundThe bulk of variability in mRNA sequence arises due to mutation—change in DNA sequence which is heritable if it occurs in the germline. However, variation in mRNA can also be achieved by post-transcriptional modification including mRNA editing, changes in mRNA nucleotide sequence that mimic the effect of mutations. Such modifications are not inherited directly; however, as the processes affecting them are encoded in the genome, they have a heritable component, and therefore can be shaped by selection. In soft-bodied cephalopods, adenine-to-inosine RNA editing is very frequent, and much of it occurs at nonsynonymous sites, affecting the sequence of the encoded protein.MethodsWe study selection regimes at coleoid A-to-I editing sites, estimate the prevalence of positive selection, and analyze interdependencies between the editing level and contextual characteristics of editing site.ResultsHere, we show that mRNA editing of individual nonsynonymous sites in cephalopods originates in evolution through substitutions at regions adjacent to these sites. As such substitutions mimic the effect of the substitution at the edited site itself, we hypothesize that they are favored by selection if the inosine is selectively advantageous to adenine at the edited position. Consistent with this hypothesis, we show that edited adenines are more frequently substituted with guanine, an informational analog of inosine, in the course of evolution than their unedited counterparts, and for heavily edited adenines, these transitions are favored by positive selection. Our study shows that coleoid editing sites may enhance adaptation, which, together with recent observations on Drosophila and human editing sites, points at a general role of RNA editing in the molecular evolution of metazoans.

Identification of Adenosine Deaminase Inhibitors by Metal-binding Pharmacophore Screening.

Adenosine deaminase (ADA) is a human mononuclear Zn2+ metalloenzyme that converts adenosine to inosine. ADA is a validated drug target for cancer, but there has been little recent work on the development of new therapeutics against this enzyme. The lack of new advancements can be partially attributed to an absence of suitable assays for high-throughput screening (HTS) against ADA. To facilitate more rapid drug discovery efforts for this target, an in vitro assay was developed that utilizes the enzymatic conversion of a visibly emitting adenosine analogue to the corresponding fluorescent inosine analogue by ADA, which can be monitored via fluorescence intensity changes. Utilizing this assay, a library of ∼350 small molecules containing metal-binding pharmacophores (MBPs) was screened in an HTS format to identify new inhibitor scaffolds against ADA. This approach yielded a new metal-binding scaffold with a Ki value of 26±1 μM.

New Linear Precursors of cIDPR Derivatives as Stable Analogs of cADPR: A Potent Second Messenger with Ca2+-Modulating Activity Isolated from Sea Urchin Eggs.

Herein, we report on the synthesis of a small set of linear precursors of an inosine analogue of cyclic ADP-ribose (cADPR), a second messenger involved in Ca2+ mobilization from ryanodine receptor stores firstly isolated from sea urchin eggs extracts. The synthesized compounds were obtained starting from inosine and are characterized by an N1-alkyl chain replacing the “northern” ribose and a phosphate group attached at the end of the N1-alkyl chain and/or 5′-sugar positions. Preliminary Ca2+ mobilization assays, performed on differentiated C2C12 cells, are reported as well.

Pharmacokinetic properties of a novel inosine analog, 4'-cyano-2'-deoxyinosine, after oral administration in rats.

4′-cyano-2′-deoxyinosine (SK14-061a), a novel nucleoside analog based on inosine, has antiviral activity against the human immunodeficiency virus type 1 that has the ability to acquire resistance against many types of reverse transcriptase inhibitors based on nucleosides. The aim of this study was to investigate the pharmacokinetics studies after its oral administration to rats. For this purpose, we first developed and validated an analytical method for quantitatively determining SK14-061a levels in biological samples by a UPLC system interfaced with a TOF-MS system. A rapid, simple and selective method for the quantification of SK14-061a in biological samples was established using liquid chromatography mass spectrometry (LC-MS) with solid phase extraction. The pharmacokinetic properties of SK14-061a in rats after oral administration were then evaluated using this LC-MS method. SK14-061a was found to be relatively highly bioavailable, is rapidly absorbed from the intestinal tract, and is then mainly distributed to the liver and then ultimately excreted via the urine in an unchanged form. Furthermore, the simultaneous administration of SK14-061a with the nucleoside analog, entecavir, led to a significant alteration in the pharmacokinetics of SK14-061a. These results suggest that the SK14-061a has favorable pharmacokinetic properties with a high bioavailability with the potential for use in oral pharmaceutical formulations, but drug-drug interactions should also be considered.

Enhancement of inosine-mediated A2AR signaling through positive allosteric modulation

Inosine is an endogenous nucleoside that is produced by metabolic deamination of adenosine. Inosine is metabolically more stable (half-life 15h) than adenosine (half-life <10s). Inosine exerts anti-inflammatory and immunomodulatory effects similar to those observed with adenosine. These effects are mediated in part through the adenosine A2A receptor (A2AR). Relative to adenosine inosine exhibits a lower affinity towards the A2AR. Therefore, it is generally believed that inosine is incapable of activating the A2AR through direct engagement, but indirectly activates the A2AR upon metabolic conversion to higher affinity adenosine. A handful of studies, however, have provided evidence for direct inosine engagement at the A2AR leading to activation of downstream signaling events and inhibition of cytokine production. Here, we demonstrate that under conditions devoid of adenosine, inosine as well as an analog of inosine 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine selectively and dose-dependently activated A2AR-mediated cAMP production and ERK1/2 phosphorylation in CHO cells stably expressing the human A2AR. Inosine also inhibited LPS-stimulated TNF-α, CCL3 and CCL4 production by splenic monocytes in an A2AR-dependent manner. In addition, we demonstrate that a positive allosteric modulator (PAM) of the A2AR enhanced inosine-mediated cAMP production, ERK1/2 phosphorylation and inhibition of pro-inflammatory cytokine and chemokine production. The cumulative effects of allosteric enhancement of adenosine-mediated and inosine-mediated A2AR activation may be the basis for the sustained anti-inflammatory and immunomodulatory effects observed in vivo and thereby provide insights into potential therapeutic interventions for inflammation- and immune-mediated diseases.

Enzymatic Interconversion of Isomorphic Fluorescent Nucleosides: Adenosine Deaminase Transforms an Adenosine Analogue into an Inosine Analogue

Adenosine deaminase (ADA), a major enzyme involved in purine metabolism, converts an isomorphic fluorescent analogue of adenosine (thA) into an isomorphic inosine analogue (thI), which possesses distinct spectral features, allowing one to monitor the enzyme-catalyzed reaction and its inhibition in real time. The utility of this sensitive fluorescence-monitored transformation for the high-throughput detection and analysis of ADA inhibitors is demonstrated.

ChemInform Abstract: Synthesis of 2′‐Substituted Inosine Analogues via Unusual Masking of the 6‐Hydroxyl Group.

Abstract2′‐Modified inosine analogues (V) and (X) are synthesized from 6‐chloropurine riboside (I) via 6‐(dimethylamino)purine (IV) or 6‐(benzyloxy)purine (VIII) intermediates.

ChemInform Abstract: One‐Pot Synthesis of 2‐Nitrooxyalkoxylated Inosine Analogues Using Cyclic Ether and Isoamyl Nitrite.

AbstractFirst examples of 2‐nitrooxyalkoxylated nucleosides are obtained by using cyclic ethers and isoamyl nitride.

Synthesis of 2′-Substituted Inosine Analogs via Unusual Masking of the 6-Hydroxyl Group

2′-Modified inosine analogs have been synthesized from 6-chloropurine riboside via 6-dimethylaminopurine or 6-benzyloxypurine intermediates. The dimethylaminopurine intermediate was obtained via an unusually facile dimethylamine transfer from dimethylformamide. Graphical :

One-Pot Synthesis of 2-Nitrooxyalkoxylated Inosine Analogs Using Cyclic Ether and Isoamyl Nitrite

An O 6 -methylguanosine derivative (1) was treated with isoamyl nitrite in the presence of a cyclic ether such as trimethylene oxide, tetrahydrofuran, or tetrahydropyran to give the corresponding 2-nitrooxyalkoxy-6-methoxypurine riboside derivatives (2a-c).

SYNTHETIC INOSINE ANALOGS IN TREATMENT OF CIN IIII

Genes products of human papillomavirus (HPV) have been identified in more then 99,7% of cervical carcinomas. Several factors may be associated with higher rates of surgical treatment failure, including highgrade CIN, large lesion size, satellite HPVrelated lesions, involved margin, persistent HPV infection. Several pharmaceutical analogs of inosine have been using about 40 years to treat broad spectrum of viral infections. Clinical and experimental studies concerning efficacy and molecular mechanism of Inosine pranobeх are characterized this product as a safe and natural immune stimulating agent with beneficial results in patients with HPV. The purpose of the study is to evaluate the effect and toxicity of the antivirus therapy of Inosine pranobeх in the complex treatment of HPV infected patients affected CIN, CC in situ and recurrence of CIN in HPVpositive women after loop conization. Surgery with the antivirus therapy of Inosine pranobeх exhibited a protective effect on the recurrence of any CIN. Inosine pranobeх is characterized as a natural immune stimulating agent without pleiotropic effects of cytokines. Our data and global clinical studies have confirmed the safety and efficacy in the treatment of CIN Inosine pranobex.

Synthesis and biological evaluation of inosine phosphonates

The 4′-phosphonomethoxy analogs of inosine and 2′,3′-dideoxyinosine were synthesized and tested for their activity against HCV and HIV, but found to be inactive. During the course of this synthetic investigation, an unexpected oxidative cleavage of the 2′,3′-bond of 2′-deoxyinosine was observed.

Adenosine binding to low-molecular-weight purine nucleoside phosphorylase: the structural basis for recognition based on its complex with the enzyme fromSchistosoma mansoni

Schistosomes are unable to synthesize purines de novo and depend exclusively on the salvage pathway for their purine requirements. It has been suggested that blockage of this pathway could lead to parasite death. The enzyme purine nucleoside phosphorylase (PNP) is one of its key components and molecules designed to inhibit the low-molecular-weight (LMW) PNPs, which include both the human and schistosome enzymes, are typically analogues of the natural substrates inosine and guanosine. Here, it is shown that adenosine both binds to Schistosoma mansoni PNP and behaves as a weak micromolar inhibitor of inosine phosphorolysis. Furthermore, the first crystal structures of complexes of an LMW PNP with adenosine and adenine are reported, together with those with inosine and hypoxanthine. These are used to propose a structural explanation for the selective binding of adenosine to some LMW PNPs but not to others. The results indicate that transition-state analogues based on adenosine or other 6-amino nucleosides should not be discounted as potential starting points for alternative inhibitors.

THE NOVEL INOSINE ANALOGUE INO-2002 EXERTS AN ANTI-INFLAMMATORY EFFECT IN A MURINE MODEL OF ACUTE LUNG INJURY

Endogenous purines, including inosine, have been shown to exert immunomodulatory and anti-inflammatory effects in a variety of disease models. The dosage of inosine required for these effects has been shown to be between 200 and 600 mg kg(-1) because of the rapid metabolism of inosine in vivo. The aim of this study was to determine whether a metabolic resistant purine analog, INO-2002, exerts anti-inflammatory effects in an animal model of acute respiratory distress syndrome. Mice challenged with intratracheal LPS (50 microg) were treated with INO-2002 (30 or 100 mg kg(-1), i.p.) in divided doses at either 1 and 12 h or at 5 and 16 h. After 24 h, bronchoalveolar lavage fluid was obtained to measure leukocyte infiltration by myeloperoxidase levels, lung edema by protein levels, and proinflammatory chemokine (macrophage inflammatory protein 1alpha) and cytokine (TNF-alpha, IL-1, and IL-6) levels. INO-2002 (30 and 100 mg kg(-1)) reduced the LPS-mediated infiltration of leukocytes and edema as evidenced by bronchoalveolar lavage fluid reduction in levels of myeloperoxidase and protein. INO-2002 also downregulated expression of the proinflammatory mediators macrophage inflammatory protein 1alpha, TNF-alpha, IL-1, and IL-6. Delaying the start of treatment by 5 h after LPS administration affected the potency of INO-2002 protective effects, with 100 but not 30 mg kg(-1) having anti-inflammatory effects. The inosine analog INO-2002 largely suppressed LPS-induced inflammation in vivo at doses lower than those needed for the naturally occurring purine inosine. These data support the proposal that purine analogs, resistant to metabolic breakdown, may represent a useful addition to the therapy of acute respiratory distress syndrome.

Synthesis and antiviral evaluation of thieno[3,4- d]pyrimidine C-nucleoside analogues of 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-adenosine and -inosine

Several thieno[3,4- d]pyrimidine derivatives, including four hitherto unknown 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro- C-nucleoside analogues of adenosine and inosine have been synthesized. When evaluated in cell culture experiments against human immunodeficiency virus, none of the tested compounds exhibited any significant antiviral effect, while two of them showed some cytotoxicity.

Synthesis and anti-HIV activity of conformationally restricted bicyclic hexahydroisobenzofuran nucleoside analogs

A chiral synthesis of a series of hexahydroisobenzofuran (HIBF) nucleosides has been accomplished via glycosylation of a stereo-defined (syn-isomer) sugar motif 16 with the appropriate silylated bases. All nucleoside analogs were obtained in 52-71% yield as a mixture of alpha- and beta-anomeric products increasing the breadth of the novel nucleosides available for screening. The structure of the novel bicyclic HIBF nucleosides was established by a single crystal X-ray structure of the beta-HIBF thymine analog 22b. Furthermore, the sugar conformation for these nucleosides was established as N-type. Among the novel HIBF nucleosides synthesized, twenty-five compounds were tested as inhibitor of HIV-1 in human peripheral blood mononuclear (PBM) cells and seven were found to be active (EC(50) = 12.3-36.2 microM). Six of these compounds were purine analogs with beta-HIBF inosine analog 22o being the most potent (EC(50) = 12.3 microM) among all compounds tested. The striking resemblance between didanosine (ddI) and 22o may explain the potent anti-HIV activity.