pinecone/core-2020-05-10-deduplication huggingface dataset:
Effect of North  Bicyclo[3.1.0]hexane 2′-Deoxy-pseudosugars on RNA Interference: A Novel Class of siRNA Modification

North bicyclo methanocarba thymidine (TN) nucleosides were substituted into siRNAs to investigate the effect of bicyclo[3.1.0]hexane 2′-deoxy-pseudosugars on RNA interference activity. Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery. We studied the effect of the TN  modification in a screen involving residue-specific changes in an siRNA targeting Renilla luciferase and we applied the most effective pattern of modification to the knockdown of murine tumor necrosis factor (TNF-α). We also showed that incorporation of TN units into siRNA duplexes increased their thermal stabilities, substantially enhanced serum stabilities, and decreased innate immunostimulation. Comparative RNAi studies involving the TN substitution and locked nucleic acids (LNAs) showed that the gene-silencing activities of TN-modified siRNAs were comparable to those obtained with the LNA modification. An advantage of the North 2′-deoxy-methanocarba modification is that it may be explored further in the future by changing the 2′-position. The results from these studies suggest that this modification might be valuable for the development of siRNAs for therapeutic applications.This research was supported by the European Union (MULTIFUN consortium), the Spanish Ministry of Education (BFU2007-63287, CTQ2010-20541), and the Generalitat the Catalunya (2009/SGR/208). This work was funded in part by the Center for Cancer Research, National Cancer Institute, NIH. M.T. acknowledges the JAE-Doc 2008 contract (CSIC, Spain) and the Juan de la Cierva contract (MICINN, Spain) for financial support.Peer reviewe
Solvent Exposure Associated with Single Abasic Sites Alters the Base Sequence Dependence of Oxidation of Guanine in DNA in GG Sequence Contexts

The effect of exposure of guanine in double-stranded oligonucleotides to aqueous solvent during oxidation by one-electron oxidants was investigated by introducing single synthetic tetrahydrofuran-type abasic sites (Ab) either adjacent to or opposite tandem GG sequences. The selective oxidation of guanine was initiated by photoexcitation of the aromatic sensitizers riboflavin and a pyrene derivative, and by the relatively small negatively charged carbonate radical anion. The relative rates of oxidation of the 5′- and 3′ side G in runs of 5′⋅⋅⋅GG⋅⋅⋅ (evaluated by standard hot alkali treatment of the damaged DNA strand followed by high resolution gel electrophoresis of the cleavage fragments) are markedly affected by adjacent abasic sites either on the same or opposite strand. For example, in fully double-stranded DNA or one with an Ab adjacent to the 5′-G, the 5′-G/3′-G damage ratio is ≥4, but is inverted (<1.0) with the Ab adjacent to the 3′-G. These striking effects of Ab are attributed to the preferential localization of the “hole” on the most solvent-exposed guanine regardless of the size, charge, or reduction potential of the oxidizing species
Iminodipropionic acid as the leaving group for DNA polymerization by HIV-1 reverse transcriptase

Previous studies have demonstrated that some selected amino monoacids and amino diacids can function as leaving groups in the polymerase-catalyzed incorporation of deoxynucleotides into DNA. Among these, the iminodiacetic acid phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) represents an interesting example, as it could overcome some of the problems observed when using L-aspartic acid as the leaving group, that is, poor chain elongation. We have now synthesized and evaluated a series of IDA-dAMP analogues that bear either an extended aliphatic chain in the amino acid function, or a phosphonic acid moiety (substituting for the carboxylic acid function). Among these compounds, the nucleotide with an iminodipropionic acid leaving group (IDP-dAMP) was identified as the best substrate; the excellent single incorporation (91 % conversion to a P+1 strand at 50 μM) was at a substrate concentration ten times lower than that used for IDA-dAMP). This nucleotide also presented improved kinetics and elongation capability compared to IDA-dAMP. The analogues with T, G, and C base moieties were also investigated for their incorporation ability with HIV-1 RT. The incorporation efficiency was found to decrease in the order A>T>G>C. The properties of the iminodipropionic acid as the leaving group surpass those of previously evaluated leaving groups; this acid will be a prime candidate for in vivo testing.status: publishe
Synthetic virus-like particles and conformationally constrained peptidomimetics in vaccine design

Conformationally constrained peptidomimetics could be of great value in the design of vaccines targeting protective epitopes on viral and bacterial pathogens. But the poor immunogenicity of small synthetic molecules represents a serious obstacle for their use in vaccine development. Here, we show how a constrained epitope mimetic can be rendered highly immunogenic through multivalent display on the surface of synthetic virus-like nanoparticles. The target epitope is the V3 loop from the gp120 glycoprotein of HIV-1 bound to the neutralizing antibody F425-B4e8. The antibody-bound V3 loop adopts a β-hairpin conformation, which is effectively stabilized by transplantation onto a D-Pro-L-Pro template. The resulting mimetic after coupling to synthetic virus-like particles elicited antibodies in rabbits that recognized recombinant gp120. The elicited antibodies also blocked infection by the neutralization sensitive tier-1 strain MN of HIV-1, as well as engineered viruses with the V1V2 loop deleted; this result is consistent with screening of V3 by the V1V2 loop in intact trimeric viral gp120 spikes. The results provide new insights into HIV-1 vaccine design based on the V3 loop, and illustrate how knowledge from structural biology can be exploited for the design of constrained epitope mimetics, which can be delivered to the immune system by using a highly immunogenic synthetic nanoparticle delivery system
Inhibition of lipopolysaccharide transport to the outer membrane in Pseudomonas aeruginosa by peptidomimetic antibiotics

The asymmetric outer membrane (OM) of Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet and phospholipid in the inner leaflet. During OM biogenesis, LPS is transported from the periplasm into the outer leaflet by a complex comprising the OM proteins LptD and LptE. Recently, a new family of macrocyclic peptidomimetic antibiotics that interact with LptD of the opportunistic human pathogen Pseudomonas aeruginosa was discovered. Here we provide evidence that the peptidomimetics inhibit the LPS transport function of LptD. One approach to monitor LPS transport involved studies of lipid A modifications. Some modifications occur only in the inner membrane while others occur only in the OM, and thus provide markers for LPS transport within the bacterial envelope. We prepared a conditional lptD mutant of P. aeruginosa PAO1 that allowed control of lptD expression from the rhamnose promoter. With this mutant, the effects caused by the antibiotic on the wild-type strain were compared with those caused by depleting LptD in the mutant strain. When LptD was depleted in the mutant, electron microscopy revealed accumulation of membrane-like material within cells and OM blebbing; this mirrored similar effects in the wild-type strain caused by the antibiotic. Moreover, the bacterium responded to the antibiotic, and to depletion of LptD, by introducing the same lipid A modifications, consistent with inhibition by the antibiotic of LptD-mediated LPS transport. This conclusion was further supported by monitoring the radiolabelling of LPS from [¹⁴C]acetate, and by fractionation of IM and OM components. Overall, the results provide support for a mechanism of action for the peptidomimetic antibiotics that involves inhibition of LPS transport to the cell surface
Programming in situ immunofluorescence intensities through interchangeable reactions of dynamic DNA complexes

The regulation of antibody reporting intensities is critical to various in situ fluorescence imaging
analyses. While such control is often necessary to visualize sparse molecular targets, the ability to
tune marker intensities is also essential for highly multiplexed imaging strategies where marker
reporting levels must be tuned to both optimize dynamic detection ranges and minimize crosstalk
between different signals. Existing chemical amplification approaches generally lack such control.
Here, we demonstrate that linear and branched DNA complexes can be designed to function as
interchangeable building blocks that can be assembled into organized, fluorescence reporting
complexes. We show that the ability to program DNA strand displacement reactions between
these complexes offer new opportunities to deterministically tune the number of dyes that are
coupled to individual antibodies in order to both increase and controllably balance marker levels
within fixed cells
An Engineered Calmodulin-Based Allosteric Switch for Peptide Biosensing

This work describes the development of a new platform for allosteric protein engineering that takes advantage of the ability of calmodulin to change conformation upon binding to peptide and protein ligands. The switch we have developed consists of a fusion protein in which calmodulin is genetically inserted into the sequence of TEM1 β-lactamase. In this approach, calmodulin acts as the input domain, whose ligand-dependent conformational changes control the activity of the β-lactamase output domain. The new allosteric enzyme exhibits up to 120 times higher catalytic activity in the activated (peptide bound) state compared to the inactive (no peptide bound) state in vitro. Activation of the enzyme is ligand-dependent-peptides with higher affinities for wild-type calmodulin exhibit increased switch activity. Calmodulin's ability to "turn on" the activity of β-lactamase makes this a potentially valuable scaffold for the directed evolution of highly specific biosensors for detecting toxins and other clinically relevant biomarkers.Chemistry and Chemical Biolog
EPR characterisation of the ferrous nitrosyl complex formed within the oxygenase domain of NO synthase.

International audienceNitric oxide is produced in mammals by a class of enzymes called NO synthases (NOSs). It plays a central role in cellular signalling but also has deleterious effects, as it leads to the production of reactive oxygen and nitrogen species. NO forms a relatively stable adduct with ferrous haem proteins, which, in the case of NOS, is also a key catalytic intermediate. Despite extensive studies on the ferrous nitrosyl complex of other haem proteins (in particular myoglobin), little characterisation has been performed in the case of NOS. We report here a temperature-dependent EPR study of the ferrous nitrosyl complex of the inducible mammalian NOS and the bacterial NOS-like protein from Bacillus subtilis. The results show that the overall behaviours are similar to those observed for other haem proteins, but with distinct ratios between axial and rhombic forms in the case of the two NOS proteins. The distal environment appears to control the existence of the axial form and the evolution of the rhombic form
Chemical and Structural Insights into the Regioversatility of the Aminoglycoside Acetyltransferase Eis

A recently discovered cause of tuberculosis resistance to a drug of last resort, the aminoglycoside kanamycin, results from modification of this drug by the enhanced intracellular survival (Eis) protein. Eis is a structurally and functionally unique acetyltransferase with an unusual capability of acetylating aminoglycosides at multiple positions. The extent of this regioversatility and its defining protein features are unclear. Herein, we determined the positions and order of acetylation of five aminoglycosides by NMR spectroscopy. This analysis revealed unprecedented acetylation of the 3′′‐amine of kanamycin, amikacin, and tobramycin, and the γ‐amine of the 4‐amino‐2‐hydroxybutyryl group of amikacin. A crystal structure of Eis in complex with coenzyme A and tobramycin revealed how tobramycin can be accommodated in the Eis active site in two binding modes, consistent with its diacetylation. These studies, describing chemical and structural details of acetylation, will guide future efforts towards designing aminoglycosides and Eis inhibitors to overcome resistance in tuberculosis.     Novel modifications of aminoglycosides:  The enhanced intracellular survival (Eis) protein of  Mycobacterium tuberculosis  can acetylate amines at two unprecedented positions on aminoglycoside antibiotics: the 3′′‐amine of kanamycin, amikacin, and tobramycin, and the γ‐amine of the 4‐amino‐2‐hydroxybutyryl group in amikacin
Methyl Yellow: A Potential Drug Scaffold for Parkinson's Disease

Parkinson's disease (PD) is an age‐related neurodegenerative disease affecting movement. To date, there are no currently available therapeutic agents which can prevent or slow disease progression. Here, we evaluated an azobenzene derivative, methyl yellow (MY), as a potential drug scaffold for PD; its inhibitory activity toward monoamine oxidase B (MAO‐B) as well as drug‐like properties were investigated. The inhibitory effect of MY on MAO activity was determined by a MAO enzyme inhibition assay. In addition, the in vitro properties of MY as a drug candidate (e.g., blood–brain barrier (BBB) permeability, serum albumin binding, drug efflux through P‐glycoprotein (P‐gp), drug metabolism by P450, and mitochondrial toxicity) were examined. In vivo effectiveness of MY was also evaluated in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) Parkinsonian mouse model. MY selectively inhibited MAO‐B in a dose‐dependent and reversible manner. MY was BBB‐permeable, bound relatively weakly to serum albumin, was an unlikely substrate for both systems of P‐gp and P450, and did not cause mitochondrial toxicity. Results from the MPTP Parkinsonian mouse model indicated that, upon treatment with MY, neurotoxicity induced by MPTP was mitigated. Investigations of MY demonstrate its inhibitory activity toward MAO‐B, compliant properties for drug consideration, and its neuroprotective capability in the MPTP Parkinsonian mouse model. These data provide insights into potential use, optimization, and new design of azobenzene derivatives for PD treatment.     Monoamine oxidase B inhibition:  We evaluated the potential use of the dye methyl yellow as a treatment for Parkinson's disease. Methyl yellow was found to be a potent inhibitor of monoamine oxidase B and protected dopaminergic neurons of mice treated with methyl yellow and toxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)
Rapid Flow-Based Peptide Synthesis

A flow-based solid-phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 min under automatic control or every 3 min under manual control is described. This is accomplished by passing a stream of reagent through a heat exchanger into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable continuous delivery of heated solvents and reagents to the solid support at high flow rate, thereby maintaining maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to that for traditional batch methods, and, in all cases, the desired material was readily purifiable by RP-HPLC. The application of this method to the synthesis of the 113-residue Bacillus amyloliquefaciens RNase and the 130-residue DARPin pE59 is described in the accompanying manuscript.MIT Faculty Start-up FundMassachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund)Deshpande Center for Technological InnovationDamon Runyon-Rachleff (Innovation Award)Sontag Foundation (Distinguished Scientist Award)C. P. Chu and Y. Lai FellowshipDaniel S. Kemp Summer FellowshipNational Institute of General Medical Sciences (U.S.). Biotechnology Training Program (Grant 5T32GM008334-25)National Institutes of Health (U.S.) (Fellowship F32GM101762
Rapid Total Synthesis of DARPin pE59 and Barnase

We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.MIT Faculty Start-up FundMassachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund)Deshpande Center for Technological InnovationDamon Runyon-Rachleff (Innovation Award)Sontag Foundation (Distinguished Scientist Award)AstraZeneca (Firm) (Distinguished Graduate Student Fellowship)Daniel S. Kemp Summer FellowshipNational Institutes of Health (U.S.). Biotechnology Training Program (5T32GM008334-25
Selective DNA-Binding by Designed Bisbenzamidine-Homeodomain Chimeras

We report the construction of conjugates between three variants of the helix 3 region of a Q50K engrailed homeodomain and bisbenzamidine minor-groove DNA binders. The hybrid featuring the sequence of the native protein failed to bind to DNA; however, modifications that increased the α-helical folding propensity of the peptide allowed specific DNA binding by a bipartite (major/minor groove) interactionMinisterio de Economía y Competitividad. Grant Numbers: SAF2010-20822-C02, CTQ2012-31341
Consolider Ingenio. Grant Number: 2010 CSD2007-00006
Xunta de Galicia. Grant Number: GRC2013/14
European Regional Development Fund
European Research Council. Grant Number: Na 340055
Ministerio de Ciencia e Innovación
Fundación Gil DávilaS
Tagging Live Cells that Express Specific Peptidase Activity with Solid-State Fluorescence

International audienceA three-component probe harnesses the extraordinary properties of a solid-state fluorophore for the detection of living cells exhibiting a particular peptidase activity. The off–on mode by which the probe operates, the bright fluorescence of the resulting precipitate, and the rapid response allow an exceptional signal-to-background ratio during microscopic imaging. A tertiary carbamate link between the spacer and phenolic fluorophore is at the heart of the probe's long-term stability. The degree of chlorination of the probe determines its response time and thus its suitability for live-cell analysis. Our probe also allows highly resolved localization of peptidase activity during gel analysis or on agar. In comparison, probes releasing soluble fluorophores demonstrate complete diffusion of the fluorescent signal. These results demonstrate the probe's potential for diverse biomedical applications, including high-fidelity flow cytometry and sensitive colony assays
Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin Protective Antigen

Antibody mimics have significant scientific and therapeutic utility for the disruption of protein–protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr-Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co-immunoprecipitation. We also observed inhibition of Bcr-Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA-mediated delivery of an affibody binder that targets hRaf-1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein–protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu.Massachusetts Institute of Technology (Startup funds)Massachusetts Institute of Technology (MIT Reed Fund)National Science Foundation (U.S.) (NSF CAREER Award (CHE-1351807))Damon Runyon Cancer Research Foundation (award)National Science Foundation (U.S.) (Graduate Research Fellowship
A Simple Fragment of Cyclic Acyldepsipeptides Is Necessary and Sufficient for ClpP Activation and Antibacterial Activity

The development of new antibacterial agents, particularly those with unique biological targets, is essential to keep pace with the inevitable emergence of drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N-acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments' antibacterial activity, the ability of fragments to activate Bacillus subtilis ClpP in vitro, and the capacity of an N-acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N-acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non-peptide activators of ClpP that can be used as antibacterial agents.National Science Foundation (U.S.)United States. National Institutes of Health (GM-101988
Targeting DNA G-Quadruplexes with Helical Small Molecules

We previously identified quinoline-based oligoamide helical foldamers and a trimeric macrocycle as selective ligands of DNA quadruplexes. Their helical structure may permit the targeting of the backbone loops and grooves of G-quadruplexes instead of the G-tetrads. Given the vast array of morphologies G-quadruplex structures can adopt, this may be a way to elicit sequence selective binding. Herein, we describe the design and synthesis of molecules based on macrocyclic and helically folded oligoamides. We tested their ability to interact with the human telomeric G-quadruplex and an array of promoter G-quadruplexes using Förster Resonance Energy Transfer (FRET) melting assay and single molecule FRET. Our results show that they constitute very potent ligands, comparable to the best reported in the literature. Their mode of interaction differs from that of traditional tetrad binders, opening avenues for the development of molecules specific for certain G-quadruplex conformations.We thank the “Cancer Research UK” for doctoral funding (SM) and “Association pour la recherche sur le cancer” for a postdoctoral fellowship (KLR). The Balasubramanian laboratory is core-funded by a programme grant from Cancer Research UK. TH acknowledges the NIH grant GM065367.This is the final version. It was first published by Wiley at http://onlinelibrary.wiley.com/doi/10.1002/cbic.201402439/abstract