A fundamentally new synthetic approach to the synthesis of 2-aminopurine has been developed. It consists in the combination of the creation of a condensed polyazotic heterocyclic tetrazolopyrimidine structure, its transformation into triaminopyrimidine, and its subsequent cyclization into 2-aminopurine. The structure of the obtained compounds was established based on spectral characteristics, and the structure of the intermediate compound 5 was established directly by X-ray diffraction analysis.

The epithelial-mesenchymal transition (EMT) is a multifunctional cell process involved in the pathogenesis of numerous conditions, including fibrosis and cancer. Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by fibroblast accumulation and collagen deposition in the lungs. The fibroblasts involved in this process partially originate from lung epithelial cells via the EMT. Evidence suggests that the EMT contributes to progression, invasion, and metastasis of various types of cancer. We screened a series of 80 compounds for the ability to interfere with the EMT and potentially be applied as a therapeutic for IPF and/or lung cancer. We identified 2-aminopurine (2-AP), a fluorescent analog of guanosine and adenosine, as a candidate in this screen. Herein, we demonstrate that 2-AP can restore E-cadherin expression and inhibit fibronectin and vimentin expression in TGF-β1-treated A549 lung cancer cells. Moreover, 2-AP can inhibit TGF-β1-induced metastasis of A549 cells. This compound significantly attenuated bleomycin (BLM)-induced pulmonary inflammation, the EMT, and fibrosis. In addition, 2-AP treatment significantly decreased mortality in a mouse model of pulmonary fibrosis. Collectively, we determined that 2-AP could inhibit metastasis in vitro by suppressing the TGF-β1-induced EMT and could attenuate BLM-induced pulmonary fibrosis in vivo. Results of this study suggest that 2-AP may have utility as a treatment for lung cancer and pulmonary fibrosis.

We previously reported that the apolipoprotein (apo) B48-carrying lipoproteins obtained from apoE knockout (apoE (-/-) ) mice, so called E(-)/B48 lipoproteins, transformed mouse macrophages into foam cells and enhanced the phosphorylation of eukaryotic translation initiation factor 2 α (eIF-2 α ). Furthermore, the eIF-2 α phosphorylation inhibitor, 2-aminopurine (2-AP), attenuated E(-)/B48 lipoprotein-induced foam cell formation. The present report studied the effect of 2-AP on atherosclerosis in apoE (-/-) mice. Our results showed that the level of food intake, bodyweight, plasma cholesterol, and triglycerides was comparable in apoE (-/-) mice treated with or without 2-AP. However, the mean size of atherosclerotic lesions in the aorta sinus as well as the surface area of the entire aorta of 2-AP-treated apoE (-/-) mice were reduced by about 55% and 39%, respectively, compared to samples from untreated control apoE (-/-) mice. In addition, the 2-AP-treated apoE (-/-) mice showed a significant decrease in glucose-regulated protein 78 (GRP78) and phosphorylated eIF-2 α in their aortic samples as compared to levels in untreated control apoE (-/-) mice. These observations suggest that endoplasmic reticulum stress is a causal mechanism for the development of atherosclerosis in apoE (-/-) mice and that therapeutic strategies can be developed for using eIF-2 α phosphorylation inhibitors, such as 2-AP, to prevent or treat atherosclerosis.

Cyclin-dependent kinase 2 (CDK2) regulates the progression of the cell cycle and is critically associated with tumor growth. Selective CDK2 inhibition provides a potential therapeutic benefit against certain tumors. Purines and related heterocycle (e.g., R-Roscovitine) are important scaffolds in the development of CDK inhibitors. Herein, we designed a new series of 2-aminopurine derivatives based on the fragment-centric pocket mapping analysis of CDK2 crystal structure. Our results indicated that the introduction of polar substitution at the C-6 position of purine would be beneficial for CDK2 inhibition. Among them, compound 11l showed good CDK2 inhibitory activity (IC50 = 19 nM) and possessed good selectivity against other CDKs. Further in vitro tests indicated that compound 11l possesses anti-proliferation activity in triple-negative breast cancer (TNBC) cells. Moreover, molecular dynamics simulation suggested the favorable binding mode of compound 11l, which may serve as a new lead compound for the future development of CDK2 selective inhibitors.

Trypanosoma cruzi infection results in debilitating cardiomyopathy, which is a major cause of mortality and morbidity in the endemic regions of Chagas disease (CD). The pathogenesis of Chagasic cardiomyopathy (CCM) has been intensely studied as a chronic inflammatory disease until recent observations reporting the role of cardio-metabolic dysfunctions. In particular, we demonstrated accumulation of lipid droplets and impaired cardiac lipid metabolism in the hearts of cardiomyopathic mice and patients, and their association with impaired mitochondrial functions and endoplasmic reticulum (ER) stress in CD mice. In the present study, we examined whether treating infected mice with an ER stress inhibitor can modify the pathogenesis of cardiomyopathy during chronic stages of infection. T. cruzi infected mice were treated with an ER stress inhibitor 2-Aminopurine (2AP) during the indeterminate stage and evaluated for cardiac pathophysiology during the subsequent chronic stage. Our study demonstrates that inhibition of ER stress improves cardiac pathology caused by T. cruzi infection by reducing ER stress and downstream signaling of phosphorylated eukaryotic initiation factor (P-elF2α) in the hearts of chronically infected mice. Importantly, cardiac ultrasound imaging showed amelioration of ventricular enlargement, suggesting that inhibition of ER stress may be a valuable strategy to combat the progression of cardiomyopathy in Chagas patients.

Adenoviruses with deletions of viral genes have been extensively studied as potential cancer therapeutics. Although a high degree of cancer selectivity has been demonstrated with these conditionally replicating adenoviruses, low levels of virus replication can be detected in normal cells. Furthermore, these mutations were also found to reduce the activity of the replicating viruses in certain cancer cells. Recent studies have shown that co-administration of chemotherapeutic drugs may increase the activity of these viruses without affecting their specificity. We constructed an adenovirus with deletions of both the E1b and the VA-RNA genes and found that replication of this virus was selective for human hepatocellular carcinoma (HCC) cell lines when compared to normal cell lines. Furthermore, we show that 2-aminopurine (2'AP) treatment selectively enhanced virus replication and virus-mediated death of HCC cells. 2'AP did not compensate for the loss of VA-RNA activities, but rather the loss of an E1b-55K activity, such as the DNA damage response, suggesting that co-administration of 2'AP derivatives that block host DNA damage response, may increase the oncolytic activity of AdΔE1bΔVA without reducing its selectivity for HCC cells.

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We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5' to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3' hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.

This article presents new data on nucleotide incision repair (NIR) activity of apurinic/apyrimidinic endonuclease Apn1 of Saccharomyces cerevisiae, which is known as a key player of the base excision DNA repair (BER) pathway, see "Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV" [1], "Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae" [2] and "Characterisation of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases" [3]. The characterization of NIR activity of wild type Apn1 and mutant form Ape1 H83A were made by denaturing PAGE analysis, and MD simulations of Apn1 complexed with DNA containing 5,6-dihydro-2'-deoxyuridine (DHU) and 2-aminopurine (2-aPu) residues. This data article is associated to the manuscript titled "Apurinic/apyrimidinic endonuclease Apn1 from Saccharomyces cerevisiae is recruited to the nucleotide incision repair pathway: kinetic and structural features" [4]. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

Here, we demonstrate that in HeLa cells, Ser317 of Chk1 undergoes phosphorylation in response to replication stress induced by hydroxyurea. We also demonstrate the existence of constitutive (interphase and mitotic) Chk1 kinase phosphorylation, the translocation of its phosphorylated form from the nucleus to cytoplasm in prometaphase as well as strong labeling of apoptotic nuclei with α-Chk1(S317) antibodies. Additionally, we show that caffeine, 2-aminopurine, staurosporine and sodium metavanadate can induce premature chromosome condensation (PCC) by the abrogation of the S-M checkpoint. Staurosporine appeared to be the most effective PCC inductor, and as in the case of the remaining inductors, the addition of hydroxyurea each time brought about an increase in the number of cells showing PCC symptoms (synergic effect). The forced premature mitosis was accompanied by an increasing index of double-strand breaks marked by the phosphorylation of histone H2AX on Ser139. Moreover, we found that the chemicals used brought about minor actin and tubulin network rearrangements that occurred following either replication stress or drug-induced cell cycle delay. At the same time, it was found that the extent of the cytoskeleton rearrangement did not hinder PCC in all its subperiods, i.e., from PCC-type prophase to PCC-type telophase.

Mitochondrial promoters of Saccharomyces cerevisiae share a conserved -8 to +1 sequence with +1+2 AA, AG or AT initiation sequence, which dictates the efficiency of transcription initiation by the mitochondrial RNA polymerase Rpo41 and its initiation factor Mtf1. We used 2-aminopurine fluorescence to monitor promoter melting and measured the kcat/Km of 2-mer synthesis to quantify initiation efficiency with systematic changes of the +1+2 base pairs to matched and mismatched pairs. We show that AA promoters are most efficient, followed by AG and then AT promoters, and the differences in their efficiencies stem specifically from differential melting of +1+2 region without affecting melting of the upstream -4 to -1 region. Inefficient +1+2 melting increases the initial NTPs Kms of the AG and AT promoters relative to AA or singly mispaired promoters. The 16-100-fold higher catalytic efficiency of AA initiation sequence relative to AG and AT, respectively, is partly due to Rpo41-Mtf1 interactions with the +1+2 non-template adenines that generate a stable pre-transcribing complex. We propose a model where the +2 base pair regulates the efficiency of initial transcription by controlling multiple steps including downstream promoter opening, +1+2 NTPs binding, and the rate of 2-mer synthesis.

Aquatic birnavirus induces mitochondria-mediated cell death, but whether connects to endoplasmic reticulum (ER) stress is still unknown. In this present, we characterized that IPNV infection triggers ER stress-mediated cell death via PKR/eIF2α phosphorylation signaling for regulating the Bcl-2 family protein expression in fish cells. The IPNV infection can induce ER stress as follows: (1) ER stress sensor ATF6 cleavaged; (2) ER stress marker GRP78 upregulation, and (3) PERK/eIF2α phosphorylation. Then, the IPNV-induced ER stress signals can induce the CHOP expression at early (6 h p.i.) and middle replication (12 h p.i.) stages. Moreover, IPNV-induced CHOP upregulation dramatically correlates to apparently downregulate the Bcl-2 family proteins, Bcl-2, Mcl-1 and Bcl-xL at middle replication stage (12 h p.i.) and produces mitochondria membrane potential (MMP) loss and cell death. Furthermore, with GRP78 synthesis inhibitor momitoxin (VT) and PKR inhibitor 2-aminopurine (2-AP) treatment for blocking GRP78 expression and eIF2α phosphorylation, PKR/PERK may involve in eIF2α phosphorylation/CHOP upregulation pathway that enhances the downstream regulators Bcl-2 family proteins expression and increased cell survival. Taken together, our results suggest that IPNV infection activates PKR/PERK/eIF2α ER stress signals for regulating downstream molecules CHOP upregulation and Bcl-2 family downregulation that led to induce mitochondria-mediated cell death in fish cells, which may provide new insight into RNA virus pathogenesis and disease.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

Current chemotherapy for acute myeloid leukemia (AML) mainly involves cytotoxic agents such as doxorubicin (DNR), mitoxantrone (Mito) or 2-aminopurine-6-thiol (6-TG). However, because these agents are relatively ineffective, discovering other more effective drugs for AML treatment would be valuable.

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3'-5' proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase-DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase.

Type 2 diabetes mellitus is characterized by insulin resistance and hypersecretion of insulin from the pancreas to compensate for decreased insulin sensitivity in the peripheral tissues. In later stages of the disease insulin-secreting beta cell degeneration commences and patients require insulin replacement therapy in order to accomplish proper regulation of their blood glucose. Endoplasmic reticulum (ER) stress in the beta cells is one of the factors contributing to this detrimental effect. Protein kinase R (PKR) is a cellular stress kinase activated by ER stress and contributing to degeneration of pancreatic islets. In order to determine whether inhibition of PKR activation by specific small molecule inhibitors of PKR ameliorates pancreatic insulin secretion capacity, we treated beta cells with two imidazole/oxindole-derived inhibitors of PKR kinase, imoxin (C16) and 2-aminopurine (2-AP), in the presence of ER stress. Our results demonstrate that PKR inhibition suppresses tunicamycin-mediated ER stress without altering the insulin production capacity of the cells. Palmitic acid-mediated suppression of insulin secretion, however, was subdued significantly by PKR inhibitor treatment through an ER stress-related mechanism. We suggest that PKR inhibitor treatment may be used to increase the insulin secretion capacity of the pancreas in later stages of diabetes.

Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Reduction of the phosphorylation state of eIF2α using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method to produce recombinant proteins from human lysates.

We performed experiments to determine the effect of PKR activation on respiratory syncytial virus (RSV) replication. We first determined that RSV infection activates PKR which induces the phosphorylation of eIF2α, resulting in the formation of host stress granules. We used RNA interference to decrease endogenous PKR levels. RSV replication was not altered in cells deficient for PKR expression. However, RSV-mediated stress granule formation was significantly reduced in PKR-knockdown cells. As an alternative method to block PKR activation, we used treatment with the kinase inhibitor 2-aminopurine (2-AP). We observed that 2-AP treatment significantly reduced viral replication. We also treated PKR-knockdown cells with 2-AP and inoculated with RSV. Under these conditions, 2-AP treatment diminished viral replication in the absence of PKR expression. These results suggest that PKR activation has a minimal effect on RSV replication and that the antiviral effect of 2-AP during RSV infection likely occurs via a PKR-independent mechanism.

Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.