121 research outputs found
Peptide dendrimer/lipid hybrid systems are efficient DNA transfection reagents: structure--activity relationships highlight the role of charge distribution across dendrimer generations.
Efficient DNA delivery into cells is the prerequisite of the genetic manipulation of organisms in molecular and cellular biology as well as, ultimately, in nonviral gene therapy. Current reagents, however, are relatively inefficient, and structure-activity relationships to guide their improvement are hard to come by. We now explore peptide dendrimers as a new type of transfection reagent and provide a quantitative framework for their evaluation. A collection of dendrimers with cationic and hydrophobic amino acid motifs (such as KK, KA, KH, KL, and LL) distributed across three dendrimer generations was synthesized by a solid-phase protocol that provides ready access to dendrimers in milligram quantities. In conjunction with a lipid component (DOTMA/DOPE), the best reagent, G1,2,3-KL ((LysLeu)8(LysLysLeu)4(LysLysLeu)2LysGlySerCys-NH2), improves transfection by 6-10-fold over commercial reagents under their respective optimal conditions. Emerging structure-activity relationships show that dendrimers with cationic and hydrophobic residues distributed in each generation are transfecting most efficiently. The trigenerational dendritic structure has an advantage over a linear analogue worth up to an order of magnitude. The success of placing the decisive cationic charge patterns in inner shells rather than previously on the surface of macromolecules suggests that this class of dendrimers significantly differs from existing transfection reagents. In the future, this platform may be tuned further and coupled to cell-targeting moieties to enhance transfection and cell specificity
Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase.
C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C-mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound. The structures indicate how the C-mannosylation sequon is recognized by this CMT and its paralogs, and how sequon binding triggers conformational activation of the donor substrate: a process relevant to all glycosyltransferase C superfamily enzymes. Our structural data further indicate that the CMTs adopt an unprecedented electrophilic aromatic substitution mechanism to enable the C-glycosylation of proteins. These results afford opportunities for understanding human disease and therapeutic targeting of specific CMT paralogs
Glycocluster Tetrahydroxamic Acids Exhibiting Unprecedented Inhibition of Pseudomonas aeruginosa Biofilms
Opportunistic Gram-negative Pseudomonas aeruginosa uses adhesins (e.g., LecA and LecB lectins, type VI pili and flagella) and iron to invade host cells with the formation of a biofilm, a thick barrier that protects bacteria from drugs and host immune system. Hindering iron uptake and disrupting adhesins’ function could be a relevant antipseudomonal strategy. To test this hypothesis, we designed an iron-chelating glycocluster incorporating a tetrahydroxamic acid and α-l-fucose bearing linker to interfere with both iron uptake and the glycan recognition process involving the LecB lectin. Iron depletion led to increased production of the siderophore pyoverdine by P. aeruginosa to counteract the loss of iron uptake, and strong biofilm inhibition was observed not only with the α-l-fucocluster (72%), but also with its α-d-manno (84%), and α-d-gluco (92%) counterparts used as negative controls. This unprecedented finding suggests that both LecB and biofilm inhibition are closely related to the presence of hydroxamic acid groups
Multivalent glycoconjugates as anti-pathogenic agents
Multivalency plays a major role in biological processes and particularly in the relationship between pathogenic microorganisms and their host
that involves protein–glycan recognition. These interactions occur during the first steps of infection, for specific recognition between host and
bacteria, but also at different stages of the immune response. The search for high-affinity ligands for studying such interactions involves the
combination of carbohydrate head groups with different scaffolds and linkers generating multivalent glycocompounds with controlled spatial
and topology parameters. By interfering with pathogen adhesion, such glycocompounds including glycopolymers, glycoclusters,
glycodendrimers and glyconanoparticles have the potential to improve or replace antibiotic treatments that are now subverted by resistance.
Multivalent glycoconjugates have also been used for stimulating the innate and adaptive immune systems, for example with carbohydrate-based
vaccines. Bacteria present on their surfaces natural multivalent glycoconjugates such as lipopolysaccharides and S-layers that can also be
exploited or targeted in anti-infectious strategie
Antimicrobial peptide dendrimers against multidrug resistant Pseudomonas aeruginosa and Acinetobacter baumannii with enhanced angiogenic effect
Multi-drug resistant bacteria represent a major public health
threat. In particular, Gram-negative pathogens such as
Pseudomonas aeruginosa are responsible for significant morbidity
and mortality in hospitals, making it urgent to develop
new classes of antimicrobials. Peptide based antimicrobials
offer an attractive opportunity to control these pathogens].
We have developed potent antimicrobial peptides with a
branched structure (peptide dendrimers) with high activity
against multi-resistant clinical isolates of P. aeruginosa and
Acinetobacter baumanii by screening a third generation peptide
dendrimer library. Our best compound G3KL is composed
of natural L-lysine and L-leucine residues (37 amino
acid residues in total) linked by amide bonds but due to the
branched topology it is stable to serum proteases in contrast
to linear peptides to red blood cells and, in contrast to linear
AMPs, stability and high activity in human serum.1 The
activity of G3KL (MIC values) is 4-8 mg/ml for a large panel
of P. aeruginosa and A. baumannii resistant strains.2 The
antimicrobial G3KL showed low toxicity to red blood cells
(MHC of 1000 mg/ml), CHO and epitelial cells. We have
also showed that peptide dendrimers such as G3KL exert direct
potent pro-angiogenic effects and can be incorporated in
biolofical bandage formulation to improve the healing process
in severe burn wounds.3
1. Combining topology and sequence design for the discovery of potent antimicrobial
peptide dendrimers against multidrug-resistant Pseudomonas aeruginosa.
Stach M, Siriwardena T N, Köhler T, van Delden C, Darbre T, Reymond J-L. Angew
Chem Int Ed. 2014, 53, 12827-31.
2. In Vitro Activity of a Novel Antimicrobial Peptide Dendrimer (G3KL) Against
Multidrug-Resistant Acinetobacter baumannii and Pseudomonas
aeruginosa. Pires J, Siriwardena T N, Stach M, Tinguely R, Kasraian S, Luzzaro
F, Leib S L, Darbre T, Reymond J-L, Endimiani A.,. Antimicrob. Agents
Chemother., 2015, 59, 7915-7918.
3. Anti-Microbial Dendrimers against Multidrug-Resistant P. aeruginosa Enhance
the Angiogenic Effect of Biological Burn-wound Bandages. Abdel-Sayed P, Kaeppli
A, Siriwardena T, Darbre T, Perron K, Jafari P, Reymond J-L, Pioletti D P, Applegate
L A, Sci. Rep., 2016, doi:10.1038/srep22020
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