Search

Two novel fluorescent peptide–based chemosensors, including A (2-amino-benzoyl-Ser-Glu-Glu-NH2) and B (2-amino-benzoyl-Ala-Glu-Pro-Glu-Ala-Glu-Pro-NH2) were synthesized and characterized by nuclear magnetic resonance (NMR) spectra. These fluorescent probes exhibited excellent selective and sensitive responses to Al3+ ions over other metal ions in aqueous buffered solutions. The limits of detection for both chemosensors towards the Al3+ ions were in the order of ∼10−7 M (A: 155 nM and B: 195 nM), which clearly indicates that these probes have significant potential for biological applications. They also displayed high binding affinity (1.3029 × 104 M−1 and 1.7586 × 104 M−1 relevant to A and B respectively). These two chemosensors are great analytical probes that produce turn-on responses upon binding to Al3+ ions through an intramolecular charge transfer (ICT) mechanism. In addition, the application of both chemosensors was examined over a wide range of pH. The fluorescent peptide–based probes and Al3+ form a 1:1 coordination complex according to the ESI-MS and Job’s plot analysis. Notably, upon addition of Al3+ to these chemosensors, a fluorescence enhancement of approximately 8-fold was observed and the binding mode was determined using NMR titration and fluorescence emission data.

3'-Fluoro-3'-deoxythymidine (FLT) was identified as one of the most potent inhibitors of human immunodeficiency virus (HIV) replication. However, FLT also showed severe toxicity so that it was abundant as a potential chemotherapeutic agent. Here, we describe various triphosphate prodrugs of FLT aiming for (a) a bypass of all phosphorylation steps needed to convert the nucleoside analogue into its triphosphate (TP) form, (b) an intracellular delivery of hydrolytically and enzymatically stable triphosphate derivatives, and (c) increasing the selectivity for HIV-RT vs three cellular DNA polymerases including the mitochondrial DNA polymerase γ. γ-Alkylated FLTTP compounds fulfill all of these requirements because these compounds proved highly resistant to dephosphorylation and showed strong selectivity for HIV-RT. Moreover, a prodrug form of these compounds proved to be nontoxic in CEM cells.

The development of nucleoside triphosphate prodrugs is one option to apply nucleoside reverse transcriptase inhibitors. Herein, we report the synthesis and evaluation of d4TTP analogues, in which the γ‐phosphate was modified covalently by lipophilic alkyl residues, and acyloxybenzyl prodrugs of these γ‐alkyl‐modified d4TTPs, with the aim of delivering of γ‐alkyl‐d4TTP into cells. Selective formation of γ‐alkyl‐d4TTP was proven with esterase and in CD4+‐cell extracts. In contrast to d4TTP, γ‐alkyl‐d4TTPs proved highly stable against dephosphorylation. Primer extension assays with HIV reverse transcriptase (RT) and DNA‐polymerases α, β or γ showed that γ‐alkyl‐d4TTPs were substrates for HIV‐RT only. In antiviral assays, compounds were highly potent inhibitors of HIV‐1 and HIV‐2 also in thymidine‐kinase‐deficient T‐cell cultures (CEM/TK−). Thus, the intracellular delivery of such γ‐alkyl‐nucleoside triphosphates may potentially lead to nucleoside triphosphates with a higher selectivity towards the viral polymerase that can act in virus‐infected cells., γ‐Alkyl‐nucleoside triphosphates were studied as DNA‐polymerase inhibitors. A strong selectivity for HIV reverse transcriptase over DNA polymerases α, β, and γ was observed. γ‐Alkyl‐nucleoside triphosphates proved highly stable in cell extracts in contrast to the parent NTPs. Lipophilic prodrugs were also prepared. High anti‐HIV activity was detected in infected CEM/TK‐deficient cells, making these compounds promising potential antivirals.

Nucleoside reverse transcriptase inhibitors (NRTIs) are widely used as antiviral and anticancer agents, although they require intracellular phosphorylation into their antivirally active form, the triphosphorylated nucleoside analogue metabolites. We report on the synthesis and characterization of a new class of nucleoside triphosphate analogues comprising a C-alkyl-phosphonate moiety replacing the γ-phosphate. These compounds were converted into bioreversibly modified lipophilic prodrugs at the γ-phosphonate by the attachment of an acyloxybenzyl (ester) or an alkoxycarbonyloxybenzyl (carbonate) group. Such compounds formed γ-C-(alkyl)-nucleoside triphosphate analogues with high selectivity because of an enzyme-triggered delivery mechanism. The latter compounds were very stable in CD4+ T-lymphocyte (CEM cell) extracts, and they were substrates for HIV-reverse transcriptase without being substrates for DNA-polymerases α, β, and γ. In antiviral assays, the excellent antiviral activity of the prodrugs that was found in CEM/0 cells was completely kept in CEM/TK- cells. The activity was improved by 3 logs as compared to the parent nucleoside d4T.

We disclose a study on nucleoside triphosphate (NTP) analogues in which the γ-phosphate is covalently modified by two different biodegradable masking units and d4T as nucleoside analogue that enable the delivery of d4TTP with high selectivity in phosphate buffer (pH 7.3) and by enzyme-triggered reactions in human CD4+ T-lymphocyte CEM cell extracts. This allows the bypass of all steps normally needed in the intracellular phosphorylation. These TriPPPro-nucleotides comprising an acyloxybenzyl (AB; ester) or an alkoxycarbonyloxybenzyl (ACB; carbonate) in combination with an ACB moiety are described as NTP delivery systems. The introduction of these two different groups led to the selective formation of γ-(ACB)-d4TTPs by chemical hydrolysis and in particular by cell extract enzymes. γ-(AB)-d4TTPs are faster cleaved than γ-(ACB)-d4TTPs. In antiviral assays, the compounds are highly active against HIV-1 and HIV-2 in wild-type CEM/O cells and more importantly in thymidine kinase-deficient CD4+ T-cells (CEM/TK-).

The broad-spectrum antiviral pseudobase T705, a fluorinated pyrazinecarboxamide, is incorporated via its triphosphate form into nascent viral RNA by viral RNA-dependent RNA polymerases. Since it mimics guanine or adenine it can act as a mutagen, whereas consecutive incorporation leads to chain termination. Here we examine the structural basis for incorporation and stalling for the case of influenza polymerase, using T1106-TP, the nucleotide form of T1105, the de-fluoro analogue of T705. We used a specially designed template that allows single T1106-MP incorporation at a defined site followed by consecutive T1106-MP incorporation and stalling four nucleotides later, as demonstrated by biochemical analysis. A high-resolution cryoEM structure of influenza A/H7N9 polymerase, stalled after transcribing this template, revealed that the entire product-template duplex has backtracked by five nucleotides. Consequently, the singly incorporated T1106-MP resides at the +1 position and forms an unexpected wobble base-pair with a U in the template. The relative stability of the canonical and wobble T1106:U base-pairs in different contexts is investigated by molecular dynamics simulations. Using a different template and influenza B polymerase we also observe stalling after double incorporation of T1106-MP and structural analysis showed again that backtracking occurs, this time by four nucleotides. These results show that, at least in early elongation, consecutive T1106-MP incorporation into the product destabilises the proximal end of the product-template duplex, promoting irreversible backtracking until a more favourable overall configuration is achieved. These results give new insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogues.

The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although dispersed, single pseudo-base incorporation is mutagenic, consecutive incorporation causes polymerase stalling and chain termination. Using a template encoding single and then consecutive T1106 incorporation four nucleotides later, we obtained a cryogenic electron microscopy structure of stalled influenza A/H7N9 polymerase. This shows that the entire product-template duplex backtracks by 5 nt, bringing the singly incorporated T1106 to the +1 position, where it forms an unexpected T1106:U wobble base pair. Similar structures show that influenza B polymerase also backtracks after consecutive T1106 incorporation, regardless of whether prior single incorporation has occurred. These results give insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product-template duplex, promoting irreversible backtracking to a more energetically favorable overall configuration.

The 35th International Conference on Antiviral Research (ICAR), sponsored by the International Society for Antiviral Research (ISAR), was held in Seattle, Washington, USA, on March 21-25, 2022 and concurrently through an interactive remote meeting platform. This report gives an overview of the conference on behalf of the society. It provides a general review of the meeting and awardees, summarizing the presentations and their main conclusions from the perspective of researchers active in many different areas of antiviral research and development. Through ICAR, leaders in the field of antiviral research were able to showcase their efforts, as participants learned about key advances in the field. The impact of these efforts was exemplified by many presentations on SARS-CoV-2 demonstrating the remarkable response to the ongoing pandemic, as well as future pandemic preparedness, by members of the antiviral research community. As we address ongoing outbreaks and seek to mitigate those in the future, this meeting continues to support outstanding opportunities for the exchange of knowledge and expertise while fostering cross-disciplinary collaborations in therapeutic and vaccine development. The 36th ICAR will be held in Lyon, France, March 13-17, 2023.

The formation of Ca

MALT1 is a key mediator of NF-κB signaling and a main driver of B-cell lymphomas. Remarkably, MALT1 is expressed in the majority of pancreatic ductal adenocarcinomas (PDACs) as well, but absent from normal exocrine pancreatic tissue. Following, MALT1 shows off to be a specific target in cancer cells of PDAC without affecting regular pancreatic cells. Therefore, we studied the impact of pharmacological MALT1 inhibition in pancreatic cancer and showed promising effects on tumor progression. Mepazine (Mep), a phenothiazine derivative, is a known potent MALT1 inhibitor. Newly, we described that biperiden (Bip) is a potent MALT1 inhibitor with even less pharmacological side effects. Thus, Bip is a promising drug leading to reduced proliferation and increased apoptosis in PDAC cells in vitro and in vivo. By compromising MALT1 activity, nuclear translocation of c-Rel is prevented. c-Rel is critical for NF-κB-dependent inhibition of apoptosis. Hence, off-label use of Bip or Mep represents a promising new therapeutic approach to PDAC treatment. Regularly, the Anticholinergicum Bip is used to treat neurological side effects of Phenothiazines, like extrapyramidal symptoms.

The antiviral drug T-705 (favipiravir) and its non-fluorinated analogue T-1105 inhibit the polymerases of RNA viruses after being converted to their ribonucleoside triphosphate (RTP) metabolite. We here compared the activation efficiency of T-705 and T-1105 in four cell lines that are commonly used for their antiviral evaluation. In MDCK cells, the levels of T-705-RTP were markedly lower than those of T-1105-RTP, while the opposite was seen in A549, Vero and HEK293T cells. In the latter three cell lines, T-1105 activation was hindered by inefficient conversion of the ribonucleoside monophosphate to the ribonucleoside diphosphate en route to forming the active triphosphate. Accordingly, T-1105 had better anti-RNA virus activity in MDCK cells, while T-705 was more potent in the other three cell lines. Additionally, we identified a fourth metabolite, the NAD analogue of T-705/T-1105, and showed that it can be formed by nicotinamide mononucleotide adenylyltransferase.

NAADP-evoked Ca

NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1-like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3-dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR.

Two novel fluorescent peptide-based chemosensors, including A (2-amino-benzoyl-Ser-Glu-Glu-NH

Nucleoside analogue reverse transcriptase inhibitors (NRTI) and nucleoside analogue monophosphate prodrugs are used in combination antiretroviral therapy (cART). The design of antivirally active nucleoside triphosphate prodrugs is a recent and an important advancement in the field of nucleoside analogue drug development. Here, we report on TriPPPro‐derivatives of nucleoside analogue triphosphates (NTPs) that comprised two different acyloxybenzyl‐masks at the γ‐phosphate of the NTP aiming to achieve the metabolic bypass. Thus, γ‐non‐symmetrically dimasked TriPPPro‐compounds (γ‐(AB,ab)‐d4TTPs) were synthesized and they proved to be active against HIV‐1 and HIV‐2 in cultures of infected wild‐type human CD4+ T‐lymphocyte (CEM/0) cells and more importantly also in thymidine kinase‐deficient CD4+ T‐cells (CEM/TK‐). From hydrolysis studies both in phosphate buffer (PB, pH 7.3) and CEM cell extracts, there was surprisingly no differentiation in the cleavage of the two acyloxybenzyl prodrug‐masks. However, if within one of the two acyloxybenzyl groups a short PEG‐type methoxytriglycol group was introduced, the “standard” acyloxybenzyl‐mask was cleaved with high preference., Masked and ready: Lipophilic nucleoside triphosphate prodrugs bearing two different bioreversible masking groups attached to the γ‐position of the triphosphate moiety were studied. The aim to use two different masking groups was to enhance the selectivity of their removal during the cleavage process in order to deliver nucleoside triphosphates intracellularly. The compounds proved to be active against HIV even in cells lacking a phosphorylating enzyme.

The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction—NAADP to NAADPH—is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.

In this minireview we describe our work on the improvement of the nucleobase analogs Favipiravir (T-705) und its non-fluorinated derivative T-1105 as influenza and SARS-CoV-2 active compounds. Both nucleobases were converted into nucleotides and then included in our nucleotide prodrugs technologies cycloSal-monophosphates, DiPPro-nucleoside diphosphates and TriPPPro-nucleoside triphosphates. Particularly the DiPPro-derivatives of T-1105-RDP proved to be very active against influenza viruses. T-1105-derivatives in general were found to be more antivirally active as compared to their T-705 counterpart. This may be due to the low chemical stability of all ribosylated derivatives of T-705. The ribosyltriphosphate derivative of T-1105 was studied for the potential to act as a inhibitor of the SARS-CoV-2 RdRp and was found to be an extremely potent compound causing lethal mutagenesis. The pronucleotide technologies, the chemical synthesis, the biophysical properties and the biological effects of the compounds will be addressed as well.

An immunostimulatory glycolipid molecule from the intestinal protozoan parasite Entamoeba histolytica (Eh) and its synthetic analogs derived from its phosphatidylinositol-b-anchor (EhPIb) previously showed considerable immunotherapeutic effects against Leishmania major infection in vitro and in vivo. Here, we describe a high content screening assay, based on primary murine macrophages. Parasites detection is based on a 90 kDA heat shock protein-specific staining, enabling the detection of several Leishmania species. We validated the assay using L. major, L. braziliensis, L. donovani, and L. infantum as well as investigated the anti-leishmanial activity of six immunostimulatory EhPIb-compounds (Eh-1 to Eh-6). Macrophages infected with dermotropic species were more sensitive towards treatment with the compounds as their viability showed a stronger reduction compared to macrophages infected with viscerotropic species. Most compounds caused a significant reduction of the infection rates and the parasite burdens depending on the infecting species. Only compound Eh-6 was found to have activity against all Leishmania species. Considering the challenges in anti-leishmanial drug discovery, we developed a multi-species screening assay capable of utilizing non-recombinant parasite strains, and demonstrated its usefulness by screening macrophage-targeting EhPIb-compounds showing their potential for the treatment of cutaneous and visceral leishmaniasis.

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. Here we show that Favipiravir exerts an antiviral effect as a nucleotide analogue through a combination of chain termination, slowed RNA synthesis and lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.

The antiviral activity of nucleoside reverse transcriptase inhibitors is often hampered by insufficient phosphorylation. Nucleoside triphosphate analogues are presented, in which the γ-phosphate was covalently modified by a non-bioreversible, lipophilic 4-alkylketobenzyl moiety. Interestingly, primer extension assays using human immunodeficiency virus reverse transcriptase (HIV-RT) and three DNA-polymerases showed a high selectivity of these γ-modified nucleoside triphosphates to act as substrates for HIV-RT, while they proved to be nonsubstrates for DNA-polymerases α, β, and γ. In contrast to d4TTP, the γ-modified d4TTPs showed a high resistance toward dephosphorylation in cell extracts. A series of acyloxybenzyl-prodrugs of these γ-ketobenzyl nucleoside triphosphates was prepared. The aim was the intracellular delivery of a stable γ-modified nucleoside triphosphate to increase the selectivity of such compounds to act in infected versus noninfected cells. Delivery of γ-ketobenzyl-d4TTPs was proven in T-lymphocyte cell extracts. The prodrugs were potent inhibitors of HIV-1/2 in cultures of infected CEM/0 cells and more importantly in thymidine kinase-deficient CD4

With an estimated number of new cases annually of approximately 1.4 million, leishmaniasis belongs to the most important parasitic diseases in the world. Nevertheless, existing drugs against leishmaniasis in general have several drawbacks that urgently necessitate new drug development. A glycolipid molecule of the intestinal protozoan parasite Entamoeba histolytica and its synthetic analogs previously showed considerable immunotherapeutic effects against Leishmania major infection. Here, we designed and synthesized a series of new immunostimulatory compounds derived from the phosphatidylinositol b anchor of Entamoeba histolytica (EhPIb) subunit of the native compound and investigated their antileishmanial activity in vitro and in vivo in a murine model of cutaneous leishmaniasis. The new synthetic EhPIb analogs showed almost no toxicity in vitro Treatment with the analogs significantly decreased the parasite load in murine and human macrophages in vitro In addition, topical application of the EhPIb analog Eh-1 significantly reduced cutaneous lesions in the murine model, correlating with an increase in the production of selected Th1 cytokines. In addition, we could show in in vitro experiments that treatment with Eh-1 led to a decrease in mRNA expression of arginase-1 (Arg1) and interleukin 4 (IL-4), which are required by the parasites to circumvent their elimination by the immune response. The use of the host-targeting synthetic EhPIb compounds, either alone or in combination therapy with antiparasitic drugs, shows promise for treating cutaneous leishmaniasis and therefore might improve the current unsatisfactory status of chemotherapy against this infectious disease.

The antiviral efficacy of many nucleoside analogues is strongly dependent on their intracellular activation by host cellular kinases to yield ultimately the bioactive nucleoside analogue triphosphates (NTP). The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. We developed a nucleoside triphosphate (NTP) delivery system (the TriPPPro approach), in which the γ-phosphate is covalently modified by two different biodegradable masking units, one is the acyloxybenzyl (AB) moiety and the other is the alkoxycarbonyloxybenzyl (ACB) group. Such compounds formed NTPs with high selectivity by an enzyme-triggered mechanism in human T-lymphocyte CEM cell extracts loosing first the AB moiety, followed by the ACB group. This enables the bypass of all steps of the intracellular phosphorylation. This approach was applied here to convert some modestly active or even inactive nucleoside analogues into powerful biologically active metabolites. Potent antiviral activity profiles were obtained depending on the lipophilicity of the TriPPPro-NTP prodrugs against HIV-1 and HIV-2 replication in cultures of infected wild-type CD4+ CEM T-cells and more importantly in thymidine kinase-deficient CD4+ T-cells (CEM/TK-). This TriPPPro strategy offers high potential for future antiviral and antitumoral chemotherapies. ispartof: JOURNAL OF MEDICINAL CHEMISTRY vol:63 issue:13 pages:6991-7007 ispartof: location:United States status: published

Adenine nucleotides (AdNs) play important roles in immunity and inflammation. Extracellular AdNs, such as adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD) and their metabolites, act as paracrine messengers by fine-tuning both pro- and anti-inflammatory processes. Moreover, intracellular AdNs derived from ATP or NAD play important roles in many cells of the immune system, including T lymphocytes, macrophages, neutrophils and others. These intracellular AdNs are signaling molecules that transduce incoming signals into meaningful cellular responses, e.g. activation of immune responses against pathogens.

Copyright © 2018, Georg Thieme Verlag. All rights reserved. We describe a short and stereospecific synthesis of different series of 1′,2′- cis -disubstituted carbocyclic nucleoside analogues. All-natural nucleobases or their precursors are coupled in a microwave-assisted Mitsunobu-type reaction with enantiomerically pure (1 R ,2 S )-2-(benzyloxymethyl)cyclopent-3-enol. By modifying the cyclopentene scaffold, our synthetic strategy gives access to a series of 1′,2′- cis -disubstituted carbocyclic nucleoside analogues of the dideoxy ( dd ), di­deoxydidehydro ( d 4) or the ribo series. The ribo series is synthesized in a more convenient way compared to a previous route. The deoxy series of 1′,2′- cis -disubstituted carbocyclic nucleoside analogues is prepared following an earlier reported approach. This synthesis involves the microwave-assisted coupling of (1 R ,2 S ,3 S )-3-(benzyloxy)-2-[(benzyl­oxy)methyl]cyclopentan-1-ol with the appropriate nucleobases. ispartof: Synthesis (Germany) vol:50 issue:11 pages:2266-2280 status: published

Herein we disclose an efficient strategy for the convergent synthesis of 1′,2′-cis-disubstituted carbocyclic ribo-nucleoside analogues. Starting from an enantiomerically pure cyclopentenol precursor, the key step for the preparation of the highly functionalized carbocyclic building block is an asymmetric dihydroxylation. Employing different variants of the Mitsunobu protocol, the condensation with all-natural nucleobases or their precursors affords a series of ribo-configured carbocyclic 1′,2′-cis-disubstituted nucleoside analogues.

The design of a bioreversibly protected lipophilic sugar nucleotide as a potential membrane-permeable precursor of adenosine diphosphate ribose (ADPR) is described. ADPR is the most potent activator of the transient receptor potential melastatin 2 (TRPM2) ion channel. Membrane-permeable, lipophilic derivatives of ADPR are of great interest as tools for study of the mechanism of TRPM2. The approach described here was based on our recently disclosed "DiPPro" and "TriPPPro" prodrug approaches developed for the intracellular delivery of nucleotides. A lipophilic, bioreversibly masked ADPR analogue containing an enzymatically cleavable 4-pentanoyloxybenzyl (PB) mask at the phosphate moiety next to the 5'-position of adenosine, together with O-acetyl groups, was prepared in high yields. Chemical and enzymatic hydrolysis studies in phosphate buffer (pH 7.3) were performed to assess chemical stability and possible (selective) enzymatic demasking of the ADPR analogue. HPLC-MS revealed that the PB group was readily cleaved enzymatically. In addition, the formation of partially deacetylated ADPR compounds and also of fully unprotected ADPR was observed.

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

With an estimated number of new cases annually of approximately 1.4 million, leishmaniasis belongs to the most important parasitic diseases in the world. Nevertheless, existing drugs against leishmaniasis in general have several drawbacks that urgently necessitate new drug development. A glycolipid molecule of the intestinal protozoan parasite Entamoeba histolytica and its synthetic analogs previously showed considerable immunotherapeutic effects against Leishmania major infection., With an estimated number of new cases annually of approximately 1.4 million, leishmaniasis belongs to the most important parasitic diseases in the world. Nevertheless, existing drugs against leishmaniasis in general have several drawbacks that urgently necessitate new drug development. A glycolipid molecule of the intestinal protozoan parasite Entamoeba histolytica and its synthetic analogs previously showed considerable immunotherapeutic effects against Leishmania major infection. Here, we designed and synthesized a series of new immunostimulatory compounds derived from the phosphatidylinositol b anchor of Entamoeba histolytica (EhPIb) subunit of the native compound and investigated their antileishmanial activity in vitro and in vivo in a murine model of cutaneous leishmaniasis. The new synthetic EhPIb analogs showed almost no toxicity in vitro. Treatment with the analogs significantly decreased the parasite load in murine and human macrophages in vitro. In addition, topical application of the EhPIb analog Eh-1 significantly reduced cutaneous lesions in the murine model, correlating with an increase in the production of selected Th1 cytokines. In addition, we could show in in vitro experiments that treatment with Eh-1 led to a decrease in mRNA expression of arginase-1 (Arg1) and interleukin 4 (IL-4), which are required by the parasites to circumvent their elimination by the immune response. The use of the host-targeting synthetic EhPIb compounds, either alone or in combination therapy with antiparasitic drugs, shows promise for treating cutaneous leishmaniasis and therefore might improve the current unsatisfactory status of chemotherapy against this infectious disease.

A chemical method for the synthesis of short strands of DNA and RNA 5'-O-triphosphates is described that makes use of the conventional coupling and oxidizing reagents that are readily available on standard DNA/RNA synthesizers. After automated solid-phase synthesis of oligonucleotides and 5'-O-detritylation, a novel cycloSal-phosphoramidite, 5-chloro-saligenyl-N,N-diisopropylphosphoramidite, was coupled to the 5'-hydroxyl group and the product oxidized. The resulting support-bonded 5'-cycloSal-oligonucleotide was reacted with pyrophosphate to yield 5'-O-triphosphorylated DNA/RNA oligonucleotides after cleavage from the support in high purity and excellent yields. The reaction sequence was adapted to be used completely on a standard automated oligonucleotide synthesizer. © 2016 by John Wiley & Sons, Inc.

T-705 (favipiravir) is a fluorinated hydroxypyrazine carboxamide that exhibits antiviral activities against a variety of RNA viruses. Given the lack of potent agents to combat these infections caused by a large number of high-impact pathogens, significant emphasis has been put on studies of the antiviral properties of T-705 and its mechanism of action. T-705 acts as a nucleobase analogue; it is therefore metabolized to the corresponding ribonucleoside triphosphate intracellularly. Herein we report a reliable synthesis of T-705-ribonucleoside as well as its 5'-monophosphate. Moreover, we disclose detailed studies on the remarkable lability of the heterocycle when attached to ribose under very mild conditions, as typically applied in biochemical studies.

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+ microdomains. Here, we identified a Ca2+ entry component involved in Ca2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Forster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+ release through RYR1, in coordination with Ca2+ entry through ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.

Adenine nucleotide (AN) 2nd messengers such as 3’,5’-cyclic adenosine monophosphate (cAMP) are central elements of intracellular signaling, but many details of underlying processes remain still elusive. Like all nucleotides, cyclic nucleotide monophosphates (cNMPs) are net-negatively charged at physiologic pH which limits their applicability in cell-based settings. Thus, many cellular assays rely on sophisticated techniques like microinjection or electroporation. This setup is not feasible for medium- to high-throughput formats, and the mechanic stress that cells are exposed to raises the probability of interfering artefacts or false-positives. Here, we present a short and flexible chemical route yielding membrane-permeable, bio-reversibly masked cNMPs for which we employed the octanoyloxybenzyl (OB) group. We further show hydrolysis studies on chemical stability and enzymatic activation, and present results of real-time assays, where we used cAMP and Ca2+ live cell imaging to demonstrate high permeability and prompt intracellular conversion of some selected masked cNMPs. Consequently, our novel OB-masked cNMPs constitute valuable precursor-tools for non-invasive studies on intracellular signaling.

We here disclose chemical synthesis of ribonucleoside 5'-monophosphate (RMP), -diphosphate (RDP), and -triphosphate (RTP) and cycloSal-, Di PPro-, and Tri PPPro nucleotide prodrugs of the antiviral pseudobase T-1105. Moreover, we include one nucleoside diphosphate prodrug of the chemically less stable T-705. We demonstrate efficient T-1105-RDP and -RTP release from the Di PPro and Tri PPPro compounds by esterase activation. Using crude enzyme extracts, we saw rapid phosphorylation of T-1105-RDP into T-1105-RTP. In sharp contrast, phosphorylation of T-1105-RMP was not seen, indicating a yet unrecognized bottleneck in T-1105's metabolic activation. Accordingly, Di PPro and Tri PPPro compounds displayed improved cell culture activity against influenza A and B virus, which they retained in a mutant cell line incapable of activating the nucleobase parent. T-1105-RTP had a strong inhibitory effect against isolated influenza polymerase, and Di PPro-T-1105-RDP showed 4-fold higher potency in suppressing one-cycle viral RNA synthesis versus T-1105. Hence, our T-1105-RDP and -RTP prodrugs improve antiviral potency and achieve efficient metabolic bypass.

Herein we describe the synthesis of lipophilic triphosphate prodrugs of abacavir, carbovir, and their 1',2'-cis-substituted carbocyclic analogues. The 1',2'-cis-carbocyclic nucleosides were prepared by starting from enantiomerically pure (1R,2S)-2-((benzyloxy)methyl)cyclopent-3-en-1-ol by a microwave-assisted Mitsunobu-type reaction with 2-amino-6-chloropurine. All four nucleoside analogues were prepared from their 2-amino-6-chloropurine precursors. The nucleosides were converted into their corresponding nucleoside triphosphate prodrugs (TriPPPro approach) by application of the H-phosphonate route. The TriPPPro compounds were hydrolyzed in different media, in which the formation of nucleoside triphosphates was proven. While the TriPPPro compounds of abacavir and carbovir showed increased antiviral activity over their parent nucleoside, the TriPPPro compounds of the 1',2'-cis-substituted analogues as well as their parent nucleosides proved to be inactive against HIV.

The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.