Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Methods Mol Biol ; 1973: 131-145, 2019.
Article in English | MEDLINE | ID: mdl-31016699

ABSTRACT

Displaying ligands in a succinct and predictable manner is essential for elucidating multivalent molecular-level binding events. Organizing ligands with high precision and accuracy provides a distinct advantage over other ligand-display systems, such as polymers, because the number and position of the ligand(s) can be accurately and fully characterized. Here we describe the synthesis of peptide nucleic acids (PNAs), which are oligonucleotide mimics with a pseudopeptide backbone that can hybridize to oligonucleotides through Watson-Crick base pair to form highly predictable and organized scaffold for organizing a ligand. The ligand(s) are covalently attached to the PNA through a squarate coupling reaction that occurs between a free amine on the ligand and a free amine appended to the pseudopeptide backbone of the PNA. In this chapter we describe the synthesis of a LKγT monomer, which ultimately yields the free amine off the backbone of the PNA, incorporation of the monomer in a PNA oligomer, and the sequential squarate coupling to conjugate the ligand.


Subject(s)
Oligonucleotides/chemistry , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/chemical synthesis , Ligands , Models, Molecular , Nucleic Acid Hybridization
2.
ACS Med Chem Lett ; 6(4): 425-9, 2015 Apr 09.
Article in English | MEDLINE | ID: mdl-25893044

ABSTRACT

Peptide nucleic acid scaffolds represent a promising tool to interrogate the multivalent effects of ligand binding to a membrane receptor. Dopamine D2 receptors (D2R) are a class of G-protein coupled receptors (GPCRs), and the formation of higher-ordered structures of these receptors has been associated with the progression of several neurological diseases. In this Letter, we describe the synthesis of a library of ligand-modified PNAs bearing a known D2R agonist, (±)-PPHT. The D2R activity for each construct was assessed, and the multivalent effects were evaluated.

3.
J Am Chem Soc ; 136(35): 12296-303, 2014 Sep 03.
Article in English | MEDLINE | ID: mdl-25116377

ABSTRACT

A programmable ligand display system can be used to dissect the multivalent effects of ligand binding to a membrane receptor. An antagonist of the A2A adenosine receptor, a G-protein-coupled receptor that is a drug target for neurodegenerative conditions, was displayed in 35 different multivalent configurations, and binding to A2A was determined. A theoretical model based on statistical mechanics was developed to interpret the binding data, suggesting the importance of receptor dimers. Using this model, extended multivalent arrangements of ligands were constructed with progressive improvements in binding to A2A. The results highlight the ability to use a highly controllable multivalent approach to determine optimal ligand valency and spacing that can be subsequently optimized for binding to a membrane receptor. Models explaining the multivalent binding data are also presented.


Subject(s)
Adenosine A2 Receptor Antagonists/pharmacology , DNA/pharmacology , Peptide Nucleic Acids/pharmacology , Receptor, Adenosine A2A/metabolism , Adenosine A2 Receptor Antagonists/chemistry , Binding Sites , DNA/chemistry , Humans , Ligands , Models, Molecular , Nanostructures/chemistry , Peptide Nucleic Acids/chemistry , Protein Binding , Receptor, Adenosine A2A/chemistry
4.
Chembiochem ; 11(16): 2302-10, 2010 Nov 02.
Article in English | MEDLINE | ID: mdl-20931643

ABSTRACT

Oligoarginine and guanidinium-rich molecular transporters have been shown to facilitate the intracellular delivery of a diverse range of biologically relevant cargos. Several such transporters have been suggested to interact with cell-surface heparan sulfate proteoglycans as part of their cell-entry pathway. Unlike for other guanidinium-rich transporters, the cellular uptake of guanidinoglycosides at nanomolar concentrations is exclusively heparan sulfate dependent. As distinct cells differ in their expression levels and/or the composition of cell-surface heparan sulfate proteoglycans, one might be able to exploit such differences to selectively target certain cell types. To systematically investigate the nature of their cell-surface interactions, monomeric and dimeric guanidinoglycosides were synthesized by using neomycin, paromomycin, and tobramycin as scaffolds. These transporters differ in the number and 3D arrangement of their guanidinium groups. Their cellular uptake was measured by flow cytometry in wild-type and mutant Chinese hamster ovary cells after the corresponding fluorescent streptavidin-phycoerythrin-Cy5 conjugates had been generated. All derivatives showed negligible uptake in mutant cells lacking heparan sulfate. Decreasing the number of guanidinium groups diminished uptake, but the three dimensional arrangement of these groups was less important for cellular delivery. Whereas conjugates prepared with the monomeric carriers showed significantly reduced uptake in mutant cells expressing heparan sulfate chains with altered patterns of sulfation, conjugates prepared with the dimeric guanidinoglycosides could overcome this deficiency and maintain high levels of uptake in such deficient cells. This finding suggests that cellular uptake depends on the valency of the transporter and both the content and arrangement of the sulfate groups on the cell-surface receptors. Competition studies with chemically desulfated or carboxy-reduced heparin derivatives corroborated these observations. Taken together, these findings show that increasing the valency of the transporters retains heparan sulfate specificity and provides reagents that could distinguish different cell types based on the specific composition of their cell-surface heparan sulfate proteoglycans.


Subject(s)
Glycosides/metabolism , Guanidine/chemistry , Heparitin Sulfate/metabolism , Animals , Biological Transport , CHO Cells , Cricetinae , Cricetulus , Dimerization , Flow Cytometry , Glycosides/chemistry , Glycosides/pharmacology , Heparitin Sulfate/chemistry , Heparitin Sulfate/pharmacology , Neomycin/chemistry , Paromomycin/chemistry , Tobramycin/chemistry
5.
Chem Commun (Camb) ; 46(30): 5542-4, 2010 Aug 14.
Article in English | MEDLINE | ID: mdl-20464029

ABSTRACT

A FRET assembly reports antibiotic affinities to two different RNA targets. A binder was labeled with a fluorophore that acts both as an acceptor for the emissive nucleoside on the bacterial A-site and a donor fluorophore for the terminally-labeled human A-site. Unlabeled drugs were used to dissociate the labeled antibiotic.


Subject(s)
Anti-Bacterial Agents/pharmacology , RNA, Ribosomal/metabolism , Bacteria/genetics , Eukaryota , Fluorescence Resonance Energy Transfer , Humans , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Ribosomal/chemistry
6.
J Am Chem Soc ; 131(48): 17605-14, 2009 Dec 09.
Article in English | MEDLINE | ID: mdl-19908830

ABSTRACT

A robust analysis and discovery platform for antibiotics targeting the bacterial rRNA A-site has been developed by incorporating a new emissive U surrogate into the RNA and labeling the aminoglycosides with an appropriate fluorescence acceptor. Specifically, a 5-methoxyquinazoline-2,4(1H,3H)-dione-based emissive uracil analogue was identified to be an ideal donor for 7-diethylaminocoumarin-3-carboxylic acid. This donor/acceptor pair displays a critical Forster radius (R(0)) of 27 A, a value suitable for an A-site-aminoglycoside assembly. Titrating the coumarin labeled aminoglycosides into the emissive A-site construct, labeled at position U1406, shows a decrease in donor emission (at 395 nm) and concurrent increase of the acceptor emission (at 473 nm). Titration curves, obtained by fitting the donor's emission quenching or the augmentation of the acceptor's sensitized emission, faithfully generate EC(50) values. Titration of unlabeled ligands into the preformed FRET complex showed a continuous increase of the donor emission, with a concurrent decrease of the acceptor emission, yielding valuable data regarding competitive displacement of aminoglycosides by A-site binders. Detection of antibiotic binding is therefore not dependent on changes in the environment of a single fluorophore, but rather on the responsive interaction between two chromophores acting as a FRET pair, facilitating the determination of direct binding and competitive displacement events with FRET accuracy.


Subject(s)
Fluorescence Resonance Energy Transfer , RNA/metabolism , Aminoglycosides/metabolism , Base Sequence , Coumarins/metabolism , Drug Design , Fluorescent Dyes/metabolism , Ligands , RNA/chemical synthesis , RNA/genetics , Time Factors , Uridine/metabolism
7.
Bioorg Med Chem Lett ; 16(9): 2522-4, 2006 May 01.
Article in English | MEDLINE | ID: mdl-16464587

ABSTRACT

(-)-Deoxypseudophrynaminol 1 possesses 43-fold greater antibacterial potency than the racemate toward Staphylococcus aureus, indicating that the (-)-enantiomer is the biologically active isomer in this assay. Comparison of the percent growth inhibition by derivatives of 1 indicates that prenylation of N8 and replacement of N1-methyl by methyl carbamate are detrimental to antibacterial potency. (-)-1 is a promising lead structure for the development of the novel hexahydropyrrolo[2,3-b]indole class of antibacterial agents.


Subject(s)
Anti-Bacterial Agents/pharmacology , Indoles/chemical synthesis , Indoles/pharmacology , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Indoles/chemistry , Indoles/classification , Microbial Sensitivity Tests , Molecular Structure , Pyrroles/chemistry , Pyrroles/classification , Stereoisomerism , Structure-Activity Relationship
SELECTION OF CITATIONS
SEARCH DETAIL
...